Received Date: May 03, 2017; Accepted Date: May 13, 2017; Published Date: May 20, 2017
Citation: Elmasry TA, Al-Ghadeer A, Al-Shaalan NH, Tousson E, El-Morshedy K (2017) Star Anise Extract Modulates Reproductive Parameters, Fertility Potential and DNA Fragmentation Induced by Growth Promoters Equigan in Rat Testes. J Appl Pharm 9:242. doi: 10.21065/1920-4159.1000242
Copyright: © 2017 Elmasry TA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Equigan is anabolic steroid that developed for veterinary use and derived from endogenous sex hormone testosterone that plays a key role in the development of male reproductive tissue as well as in puberty and spermatogenesis. The current study aimed to investigate the possible prophylactic effect of Star Anise Extracts (SAE) on the toxicity of rat testes, sexual hormones alternations, sperm count, sperm abnormalities and testicular DNA damage by Equigan. Forty male adult rats were equally divided into four groups. First Control group, second group were rats receive orally SAE. 3rd group include rats that injected intramuscularly with Equigan while 4th group were co-treated Equigan with SAE. Food and fluid intakes, relative body weight, potassium, chloride, phosphorous, non-progressive and immotile sperms were significantly increased in Equigan group as compared to control group. In contrast; relative testes weight, sodium, magnesium, total calcium, testosterone, FSH, LH, PRL, sperm count, progressive motility and viability showed a significant decrease in Equigan as compared to control groups. Relative weight of epididymis, seminal vesicles and prostates and serum calcium ions didn’t change significantly in different studied groups. Co-administration of SAE with Equigan improved the sexual toxicity, electrolyte alternations, sperm count, abnormalities and DNA damage induced by Equigan.
Anabolic steroid; Equigan; Testis; Sperm marphometery; Star anise; Rat
Anabolic-androgenic steroids are defined as synthetic derivatives of the endogenous sex hormone testosterone that plays a key role in the development of male reproductive tissue (such as the testis and prostate) as well as in puberty, sexual behaviour, spermatogenesis and plays promoting secondary sexual characteristics such as increased muscle, bone mass and hair growth [1-6]. Anabolic steroids are forbidden for meat production and human uses in KAS and most countries worldwide due to their undesirable effects included blood and cardiovascular disorders, liver dysfunction, kidney disease, tendon damage, testicular problems, psychiatric and behavioural disorders in both sexes as well as other problems on human body [7-18].
Equigan is anabolic steroid that developed for veterinary use to improve the food producing animal growth rate through promoting protein synthesis and also Equigan have been reclassified as Schedule III drugs and in addition it classified as class 2A (growth promotors– steroids according to the International Agency for Research on Cancer; ISRC), as a probable human carcinogen with a high carcinogenic index [19-21]. Recently, in order to get a better physical performance and muscular appearance, many young people seek resources that, in most cases, can bring harm to health, such as the indiscriminate use of Equigan.
Star anise (Illicium verum) is aromatic evergreen trees that grows in China and Vietnam and are traditionally used in the treatment of stomach aches, insomnia, vomiting, inflammation and rheumatic pain [22,23].
Star anise crude extracts possess wide pharmacological properties, such as an antioxidant by reducing free radical production and lipid peroxidation and antimicrobial, additionally, it considered as the main source of shikimic acid, which is a primary ingredient of the Tamiflu drug [24,25]. Therefore, the aim of the current study was to investigate the possible prophylactic effect of Star Anise Extracts (SAE) on the toxicity of the rat testes, sexual hormones alternations, sperm count, sperm abnormalities and testicular DNA damage by Equigan.
Chemicals and reagents
Equigan® vial was obtained from Laboratories Tornel Co., (S.A. Mexico). Each vial containing oily solution (50 mg/ml vehicle).
Healthy male albino rats (weighting 180-200 g and 12-14 weeks age), supplied from the accredited breeding and experimental laboratory (Tanta Alpha Centre, Egypt) were used for this study. The animals had free access to water. Rats were monitored closely during the treatment period (12 weeks). The food intake, fluid intake and body weight were recorded weekly throughout the experimental period. All the experiments were designed and conducted according to the ethical norms approved by the Ethical Committee of National Research Center. The experimental procedures were approved by the Committee of Ethics in the Use of Experimental Animals –ENRC (Protocol No. 039/2008).
After 2 weeks of acclimatization, rats were assigned to 4 groups (10 animals each).
he rats will not receive any treatment.
Star anise group
The rats will receive orally star anise extract (SAE) by stomach tube (100 mg/kg BW/twice a week) for 12 weeks .
The rats will injected intramuscular with Equigan (5 mg/kg BW/ week) for 12 weeks .
Treated equigan with SAE group
The rats will receive intramuscular injections of Equigan at (5 mg/Kg body weight\week) with oral SAE (100 mg/kg body weight/ twice a week) together for 12 weeks.
At the end of the experimental period, rats from each group were euthanized with anesthetic ether and subjected to a complete necropsy after 10-12 h of fasting. Blood samples were collected from the inferior vena cava of each rat in non-heparinized glass tubes. Blood serum was separated; collected and stored at -18°C. Testes, epididymis, seminal vesicles and prostates were removed, carefully cleaned in cold saline and weighed.
Total calcium levels in serum were determined by using commercial kits (Bicon Co, Germany) . While magnesium levels in serum were determined by using commercial kits (BioMérieux Co, France) . Serum potassium, sodium, calcium and chloride ion levels in were determined by using commercial kits (Sensa core electrolyte, India).
Prolactin levels in rat serum were assayed by using the ADVIA Centaur XP system (two-site sandwich immunoassay using direct chemiluminometric technology; Vidas, France). The First antibody, in the Lite Reagent, is a polyclonal goat anti-prolactin antibody labeled with acridinium ester. The second antibody, in the Solid Phase, is a monoclonal mouse anti-prolactin antibody, which is covalently coupled to paramagnetic particle.
Luteinizing Hormone (LH) levels in rat serum were assayed by using the ADVIA Centaur XP system (two-site sandwich immunoassay using direct chemiluminometric technology; Vidas, France). The First antibody is a monoclonal mouse anti-LH antibody labeled with acridinium ester. The second antibody, in the Solid Phase, is a monoclonal mouse anti-LH antibody, which is covalently coupled to paramagnetic particles.
Follicle Stimulating Hormone (FSH) levels in serum was assayed by using the ADVIA Centaur XP system (two-site sandwich immunoassay using direct chemiluminometric technology; Vidas, France). The first antibody is a polyclonal sheep anti-FSH antibody bound to monoclonal mouse anti-FSH antibody, coupled to para-magnetic particles in the solid phase.
Total testosterone concentrations in serum were assayed by using the ADVIA Centaur XP system (two-site sandwich immunoassay using direct chemiluminometric technology; Vidas, France). Testosterone in the rats sample competes with acridinium ester-labeled testosterone in the reagent for a limited amount of polyclonal rabbit anti-testosterone antibody bound to monoclonal mouse anti-rabbit antibody, coupled to para-magnetic particles in the solid phase.
Sperms morphometric analysis
Left caudal part of the epididymis were carefully separated from each testis, finely minced in 5 ml of Hanks’ buffered salt medium, and incubated at room temperature for 15 min to provide the migration of all spermatozoa from epididymal tissue to fluid. To evaluate sperm counting, spermatozoa motility parameters and sperm morphology a computer assisted semen analysis (CASA System; MiniTüb, Germany) with Olympus microscope (Olympus, Tokyo, Japan) was used .
Assess the spermatozoa morphological abnormalities, after semen collection, samples were fixed with Hancock’s solution, smears were prepared on a slide and air-dried and made permanent smeared slide was stained with 1% eosin Y and 5% nigrosin. Morphological sperm defects were evaluated and examined on Olympus microscope using 400X magnifications. A total of 200 spermatozoa from each rat were examined and individually scored normal or abnormal, according to the strict sperm morphology criteria .
DNA damage in testis from different groups was tested by using the diphenylamine . The percentage of DNA fragmentation in each sample was expressed by the following formula:
% DNA fragmentation=(O.D. Supernatant/O.D. Supernatant+O.D. pellet) × 100
Where O.D. is optical density.
Data were expressed as mean values ± SE and statistical analysis was performed using one way ANOVA to assess significant differences among treatment groups. The criterion for statistical significance was set at p<0.01 for the biochemical data. All statistical analyses were performed using SPSS statistical version 21 software package (SPSS® Inc., USA).
Table 1 showed that food and fluid intakes and relative body weight (RBW) showed a significant increase in Equigan group as compared to control and SAE groups. Meanwhile, relative testes weight showed a significant decrease in Equigan group as compared to control and SAE groups. On the other hand; the relative weight of epididymis, seminal vesicles and prostates didn’t change significantly in different studied groups.
|Water intake (ml/rat/day)||35.5# ± 1.98||35.9# ± 2.50||42.4* ± 1.44||39.8* ± 2.62|
|Food intake (g/rat/day)||13.4# ± 0.57||13.8# ± 0.95||19.0* ± 0.58||16.9*# ± 0.44|
|RBW (g/100 g)||26.5# ± 1.03||29.8# ± 0.89||47.2* ± 2.21||36.8*# ± 1.25|
|RTW (g/100 g)||1.34# ± 0.045||1.37# ± 0.029||1.09* ± 0.032||1.26*# ± 0.081|
|REW (g/100 g)||0.332 ± 0.046||0.335 ± 0.022||0.330 ± 0.018||0.335 ± 0.048|
|RPW (g/100 g)||0.310 ± 0.018||0.316 ± 0.015||0.309 ± 0.011||0.311 ± 0.027|
|RSVW (g/100 g)||0.44 ± 0.076||0.43 ± 0.061||0.42 ± 0.045||0.42 ± 0.07|
|Value represents mean ± SE of 10 rats. Significant difference from the control group at *p<0.05. Significant difference from Equigan group at #p<0.05. Relative testes weights (RTW), relative Epididymis weights (REW), relative seminal vesicles weights (RSVW) and relative prostates weights (RPW). Relative organ weight=Organ weight × 100
Table 1: Changes in the water intake, food intake, Relative Body Weights (RBW) and relative organ weights (g/100 g body weight) of sex organs in different groups.
Effect of equigan and SAE on electrolytes alterations
It was clearly evident from Table 2 that serum sodium, magnesium and total calcium levels were a significantly decrease in Equigan group as compared to control and SAE groups. In contrast; serum potassium, chloride and phosphorous were a significantly increase in Equigan group as compared to control and SAE groups. On the other hand; a significant increase in serum sodium, magnesium and total calcium levels in co-treatment Equigan with SAE group when compared with Equigan group, while a significant decrease in serum potassium, chloride and phosphorous levels in co-treatment Equigan with SAE group when compared with Equigan group. In addition, serum calcium ions didn’t change significantly in different studied groups (Table 2).
|Na+ (mmol/l)||135.4 ± 8.435b||136.9 ± 5.266b||127.1 ± 4.519a|
|K+ (mmol/l)||4.365 ± 0.198b||4.410 ± 0.124b||5.633 ± 0.138a|
|Ca++ (mmol/l)||1.261 ± 0.094||1.248 ± 0.055b||1.211 ± 0.032|
|Ca total (mg/dl)||10.54 ± 0.329b||10.61 ± 0.541b||9.46 ± 0.187ab|
|Cl- (mmol/l)||100.3 ± 4.35b||102.0 ± 8.81b||119.1 ± 5.55a|
|Mg (mg/dl)||2.17 ± 0.055b||2.31 ± 0.028b||1.13 ± 0.019a|
|Ph (mg/dl)||4.94 ± 0.18b||4.87 ± 0.24b||7.43 ± 0.41a|
|Value represents mean ± SE of 10 rats, Significant difference from the control group at *p<0.05. Significant difference from Equigan group at #p<0.05|
Table 2: Changes in electrolyte levels in different groups.
Effect of equigan and SAE on reproductive hormones
It was clearly evident from Figures 1 and 2 that testosterone, FSH, LH and PRL levels were a significantly decrease in Equigan group as compared to control and SAE groups. In contrast; a significant increase in total testosterone, FSH, LH and PRL in co-treatment Equigan with SAE group when compared with Equigan group.
Semen characteristics was affected after Equigan injection, were a significant depression in the sperm count, motility and viability in Equigan group as compared to control group (Tables 3 and 4). In contrast; a significant increase in sperm count, motility and viability in co-treatment Equigan with SAE group when compared with Equigan group.
|Sperm count (million/ml)||81.9# ± 8.52||86.1# ± 7.35||53.66* ± 4.21||74.98#* ± 8.45|
|Vitality (%)||79.85# ± 3.74||72.05# ± 3.11||54.19* ± 3.46||71.55#* ± 5.65|
|Each reading represents Mean ± SEM of 10 rats. The significance of difference was checked by t test and multiple comparison Dunnett test (compare all vs control) using a computer program graph pad (Instat software Inc.). Significant difference from the control group at *p< 0.05. Significant difference from Equigan group at #p< 0.05|
Table 3: Changes in the epididymal sperm count and vitality in the different groups under study.
|Total motility (PR+NP)||70.05# ± 2.69||71.35# ± 2.45||58.28* ± 3.48||66.09#* ± 3.39|
|Progressive motility (PR)||47.65# ± 2.35||51.23# ± 4.37||33.36* ± 1.75||44.55# ± 2.15|
|Non Progressive (NP)||22.40# ± 1.15||20.12# ± 1.07||24.92* ± 1.33||21.54# ± 1.62|
|Immotile (IM)||29.95# ± 0.97||28.65# ± 1.91||41.72* ± 2.27||33.91#* ± 2.29|
|Each reading represents Mean ± SEM of 10 rats. The significance of difference was checked by t- test and multiple comparison Dunnett test (compare all vs control) using a computer program graph pad (Instat software Inc.). Significant difference from the control group at *p< 0.05. Significant difference from Equigan group at #p< 0.05|
Table 4: Changes in the epididymal sperm total motility (PR+NP).
Table 4 showed the changes in the epididymal sperm total motility (PR+NP), PR: Progressive Motility; NP: Non-Progressive; IM: Immotile; % in the different groups under study in the different groups under study. Progressive motility were a significantly decrease in Equigan group as compared to control and SAE groups. In contrast; non progressive and immotile sperms were a significantly increase in Equigan group as compared to control and SAE groups. In contrast; non progressive and immotile sperms showed a significant decrease in co-treatment Equigan with SAE group when compared with Equigan group.
Figure 3 show the sperm head abnormalities of rats treated with Equigan, the percentage of sperm with head defect (no head) has been increased significantly the Equigan group as compared with control group; in addition, there is a significant increase in other head defect like amorphous, banana shape, hook less head, pin head sperms in Equigan group as compared with control group. In contrast; a significant decrease in sperm head abnormalities in co-treatment Equigan with SAE group when compared with Equigan group (Figure 3). The percentage of sperms with bent mid piece was significantly increased in control rats group as compared with Equigan group while curved mid-piece was significantly increased in Equigan rats group as compared with control group.
Figure 3: Microphotographs illustrating morphologically normal sperm (a) and various sperm defects (b-k). b) Sperm agglutination; c) Fused sperm; d) Bent head; e) Double head; f) Hook less head (white arrow) and hook head (black arrow); g) Amorphous head (white arrow) and banana head (black arrow); h) Tailless head; I) Sperms with bent mid piece; j) Coiled tail; k) Looped tail.
Also; Figure 3 show the sperm tail abnormalities or tail defect (as headless, bent, curved, coiled and looped tails) of rats treated with Equigan were significantly increased in as compared with control group. In contrast; a significant decrease in sperm tail abnormalities in co-treatment Equigan with SAE group when compared with Equigan group.
In the present study, we found that; intramuscular injections of Equigan to rats resulted in a significant increase in testicular DNA fragmentation (P<0.001 vs. control group). Treatment with star anise extract inhibited the cellular damage significantly (P<0.001) (Figure 4).
Anabolic steroid has dual effects on humans (directly and indirectly) directly as intramuscular injection to build muscles and indirectly as through consuming meat of animals that were treated with Equigan . The present results revealed that intramuscular injection of the anabolic steroid Equigan to male rats evoked a significant elevation in the food intake, fluid intake and relative body weight rate and a significant depletion in the relative testes weight in Equigan group as compared to control group while the relative weights of epididymis, seminal vesicles and prostates didn’t revealed any changes between the different groups under study. Similar findings were reported by Saleh and Waded ; El-Moghazy et al. ; Tousson et al.  and Mohammed et al.  who reported that the body weight and total protein concentrations in male rabbits were significantly increased after boldenone injections. Also, Silcox et al.  reported a significant increase in weight gain of lambs after trenbolone acetate injections. In contrast, Cannizzo et al.  find no statistically relevant difference between different groups of veal calves treated with boldenone. The results are not agreement with Oda and. El-Ashmawy who reported that boldenone had no significant effect on the body weight and body weight gain while the testes and epididymis weights were decreased significantly after boldenone injections in male rabbits . This effect could be attributed to Equigan acting upon the androgen receptors in anabolic-responsive tissues promoting the body tissue building processes due to increased protein synthesis.
Magnesium ions regulate over 300 biochemical reactions in the body through their role as enzyme co-factors. Chloride ion is an essential electrolyte located in all body fluids responsible for maintaining acid/base balance and transmitting nerve impulses. Also, chloride ion is a chlorine anion that forms the negatively charged part of certain salts, including sodium and hydrogen chloride salts. The present work showed a significant decrease in sodium, magnesium and total calcium levels in Equigan as compared to control groups while a significant increase in potassium, chloride and phosphorous in Equigan as compared to control groups. In contrast, Treatment with SAE improved this alteration in electrolytes. These results agree with Pitts and Davis who reported that an evidence- based analysis of anabolic steroids as performance enhancers in horses . Also, the current results agree with Barakat et al.  These results agree with those obtained in humans where administration of boldenone undecylenate to elderly human subjects, also agree with O'Connor et al. who reported that evaluation of boldenone undecylenate as an anabolic agent in horses . The current results revealed that; the level of total testosterone, FSH, LH and PRL hormone dramatically dropped (P<0.05) in Equigan treated rats group compared with the control group. But rats treated with Equigan and star anise extract (Equigan+SAE) significantly (p<0.05) increased the level of total testosterone, FSH, LH and PRL hormone compared with Equigan treated rats. This remarkable reduction of sexual hormones which were found in the current study might be explained by severe damages, which Equigan exerted on leydig and sertoli cells by increased generation of free radicals is one of the possible mechanisms involved in anabolic-androgenic steroid-induced Leydig cell degeneration. In agreement with our findings, Purkayastha and Mahanta who reported that; a significant decrease in the FSH, LH and testosterone levels after Nandrolone Decanoate injections in mice . These results agree also with Mohammed et al. ; Oda and. El-Ashmawy  and Thabet et al.  who reported that; a significant reduction in the total testosterone after boldenone injections. In contrast, administration of testosterone alone to bull calves did not induce any variation in testosterone . Also, Shimomura et al. showed that; a significant decrease in testosterone levels after ethinylestradiol alone treatment . Not similar findings were reported by Urhausen et al.; Takahashi et al. and Gabr et al. who reported that; significant increase in serum testosterone levels in treated groups with anabolic-androgenic steroid when compared with control group [42-44].
The obtained results indicate that; intramuscular injection of rats with Equigan adversely affects spermatogenesis, sperm motility and sperm count suggesting that anabolic-androgenic steroid hormone might play an important role not only in controlling normal testicular development, but also in maintaining normal testicular function and spermatogenesis. Suppression of sperm output is attributed to the degree of inhibition of germ cell development which is related to the degree of FSH, LH and testosterone suppression . This could explain decreased sperm concentration, motility and abnormal morphology induced by Equigan administration to rats. The decrease in the sperm count after Equigan intramuscular injection may be due to decreased level of intravesicular testosterone, because testosterone levels is directly linked to spermatogenesis. Our results agree with Tousson et al.  who reported that; boldenone undecylenate affects spermatogenesis by inhibiting the nucleic acid synthesis of germ cells and redaction in gonadotropin secretion causes atrophy of the testes. Our results were similar to those reported by Brown  and Ciocca  in athletes and Cannizzo et al.  in veal calves. Also, our results agree with Oda and. El-Ashmawy  and Thabet et al.  who reported that; a significant reduction in the sperm motility and count after boldenone injections while no abnormalities were detected in the sperm morphology after boldenone injections. According to results from the CASA analysis, different motility patterns were identified after Equigan injection. The decrease in normal sperm morphology after Equigan injection was linked to significantly increased sperm with abnormal heads. The decrease in sperm count, the increase in dead and abnormal sperm of rats may occur due to increase free radical formation initiating germ cell apoptosis and subsequent male infertility. On the other hand, the presence of star anise with Equigan improved the sperm motility. However, the incidence and severity of testicular and epididymal lesions in rats pre-treated with star anise was considerably ameliorated when compared to that in the Equigan group. Therefore, star anise attenuated the Equigan-induced testis and epididymis damages .
Two main mechanisms are known by which reactive oxygen species (ROS) can causes infertility. The first mechanism, ROS can damage the sperm membrane, which in turn, reduces sperm motility and their ability to fuse with the oocyte, while the second mechanism, ROS directly damage sperm DNA. The current results revealed that, rat injections with Equigan increased testicular DNA fragmentation, indicated that Equigan is able to induce testicular apoptosis. Our results were in agreement with Meseguer et al. who reported that; DNA damage of sperm cell decrease the quality of the sperm, increase the abnormalities and cause fertilization problems since spermatozoa requires intact DNA during fertilization process . In contrast; SAE improved the testicular DNA damage and this results agree with Padmashree et al.  and Dinesha et al.  who studies the antioxidant and DNA protectant activities of star anise aqueous extracts. So, it is therefore possible that SAE could scavenge free radicals and produce beneficial effects against Equigan damage in testis.
This work was supported by the Deanship of Scientific Research (DSR), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia, under grant No.37-K-189. The authors therefore, gratefully acknowledge the DSR technical and financial support.