Chainarong Tocharus, Ph.D.,1 Dawan Shimbhu, Ph.D.,2 Jiraporn Tocharus, Ph.D.3 and Arampa Ruchiratanti-angkoor, M.D.1
1Department of Anatomy, Faculty of Medicine, Chiang Mai University, 2Department of Biochemistry, Faculty of Medical Science, Naresuan University, 3Department of Physiology, Faculty of Medicine, Chiang Mai University
To investigate the long term effects of ethanolic extracts of Butea Superba Roxb. root on sperm number and fertility, sperm quality and histology of testis in male hamsters.
Male hamsters (100-120 g) were divided into four equal groups: one control group and three different treatment groups. The control group received distilled water orally each day for 6 months. The treatment groups were fed with alcoholic extract suspensions of B. superba root at doses of 0.1, 1 or 10 mg/kg BW/day for 6 months. The fertility of sperm was assessed by deter-mining the number of 2-cell embryos collected from super-ovulated female hamsters after mating. The quality of sperm in male hamsters treated with B. superba was evaluated by counting the number of sperm, and the number of intact acrosomes on the sperm head. Histopathologic study of the testes was performed to evaluate the number of spematogonia.
After six months of daily oral treatment with root extract, the sperm count was increased in the treated male hamsters in a dose-dependent manner. The number of 2-cell embryos in super-ovulated females, after mating with treated males, was significantly greater in B. superba-treated groups than in the control group. There was no change between the treated and control group in the percentage of sperm with acrosome. No pathology of reproductive organs was observed.
B. superba root extract increases spermatogenesis among male hamsters, without affecting fertility and acrosome integrity, after 6 months of oral treatment.
Keywords: acrosome; Butea superba Roxb., fertilization, sperm fertility, triple stain
Butea superba Roxb. (known locally as Kwao Khrua Dang) is found in northern Thailand as a climbing tree with long tuberous roots and red sap . Products from this plant have been used by humans as a physical and mental tonic and for the prevention of age-related health problems
Thai traditional medicine uses B. superba as a rejuvenating herb for men. Compounds extracted from B. superba have been effective in vitro in inhibiting cAMP phosphodiesterase; the mechanism that plays an important role in penile erection . Previous studies have shown that ethanolic extract of B. superba induced penile erection by increasing intracavernous pressure with the maximum effective dose of 1 mg/kg, and also relaxed the corpus cavernosal smooth muscle in rat models . The clinical trial of B. superba powder in Thai males showed effective treatment of erectile dysfunction . The study on chronic toxicity of B. superba examined its effects on body weight, food consumption, rela-tive organ, hematology, blood chemistry and histopathology . Although there have been a considerable number of investigations into the effects of reproductive toxicants on the histology of the testis and ovary, there are no reports on damage to the fertility of sperm. Fertilization in vivo requires adequate numbers of spermatozoa with normal morphology and motility to be ejaculated [7, 8]. Since spermatogenesis involves a complex process of cellular development, im-pairment at any stage may lead to a reduction in fertility. Spermatogenesis has been shown to be susceptible to damage from a variety of chemical agents .
The aim of this study was to examine the effect on fertility of long-term treatment of alcoholic extract of B. superba root, as shown by sperm concentration and acrosome integrity of sperm, in correlation to the testicular architecture in hamsters.
Male hamsters (100-120 g) were obtained from the National Laboratory Animal Center, Mahidol University, Salaya, Nakornpathom, Thailand. They were housed un-der a 12:12 h light-dark cycle and maintained at 25 ± 3 °C. The experimental protocol was approved by the Committee on Animal Experimentation, Faculty of Medical Science, Naresuan University.
Preparation of root extract
Tuberous roots of B. superba were collected from Phrae province, Thailand, in November, 2006. This plant was identified by the Botany and Weed Science Division, Department of Agriculture, Kasetsart University, Bangkok, with the sample voucher code of MFLU 310. The roots were sliced into small pieces and extracted with ethanol. The ethanolic extract was evaporated under reduced pressure at 45 oC, using a rotary evaporator, and stored at -20 °C until use.
Treatment of hamsters with B. superba root extract Male hamsters were divided randomly into four groups of six hamsters per group. The control group received distilled water orally, whereas the treatment groups were fed with 1-mL suspension of B. superba root extract at doses of 0.1, 1 and 10 mg/kg BW/day through 17- gauge tubing for 6 months.
Measurement of body and reproductive organ weight
Body weight was recorded before treatment, at weekly intervals during treatment and at the end of treatment. Testes, epididymis, seminal vesicle and prostate were excised and weighed after removal of surrounding adipose tissue.
Epididymal sperm counts were determined, as described previously [10, 11]. Briefly, the cauda epididymis was re-moved, cut and weighed, then finely minced with scissors to release the epididymal content into a 35-mm Petri dish containing 2 mL of Tyrode’s solution (pH 7.4). The sperm was counted using a Neubauer hemocytometer.
The acrosome status was assessed by triple stain proce-dure . A 200 μL sample from each treatment suspen-sion was incubated with an equal volume of TALP-HEPES (HEPES 10 mM, NaCl 127 mM, KCl 3.16 mM, CaCl2.2H2O 2.0 mM, MgCl2.6H2O 0.5 mM, Na lactate 10 mM, NaH2PO4. H2O 0.35 mM, NaHCO3 2.0 mM, Glucose 5.0 mM) con-taining 2% Trypan Blue at 37 °C for 10 min, and then cen-trifuged at 200 g for 10 min. The pellet was resuspended with 0.5 mL of TALP- HEPES. Three smears were made from each sample and fixed with 2.5% glutaraldehyde for 1 h. The fixed sperm was air-dried and stained overnight with 0.8% Bismark Brown for 5 min at 40 °C, and 0.8% Rose Bengal for 30 min at 24 °C. Live and dead sperm were dis-tinguished in this procedure by staining with Trypan Blue, in the presence of acrosome on the sperm head resulting in uptake of Rose Bengal at the sperm tip. Slides were evalu-ated by counting 400 cells, using bright field microscopy, and results were expressed as percentage of sperm with an intact acrosome.
Testicular histology study
After the hamsters were sacrificed, their testes were dissected, cut into small slices and fixed in a 10% formalde-hyde buffer for 24 h. The tissue was washed free of fixative, dehydrated in alcohol series, and then embedded in paraffin. Tissue sections of 5-μm thickness were prepared and placed on glass slides. The sections were stained with hematoxylin and eosin and mounted in a mounting medium. The slides were observed under a light microscope for qualitative changes in the seminiferous tubules.
A male was mated with two super ovulated females at the end of the six-month treatment period. Super ovulation was achieved by intraperitoneal injection of 25 μL of pregnant mare serum gonadotropin, followed 72 h later by administration of 25 μL of human chorionic gonadotropin. In the following morning, presence of vaginal plug and sperm in the vaginal smear was considered positive indices of mating. After 48 h, the mated females were sacrificed and their oviducts removed. The 2-cell stage embryos were recovered by flushing the oviducts with Dulbecco’s phosphate buffered saline and counted under a microscope.
Statistical comparison between different treatment results was analyzed by one-way analysis of variance followed by the LSD test. Differences with a p-value of 0.05 or less were considered statistically significant.
Effect of B.superba root extract on body and reproductive organ weight
After 6 months of daily oral treatment with B. superba root extract, the mean body weight in all of the treatment groups did not differ significant-ly from that in the control group. Similarly, the mean weight of reproductive organs (testes, epi-didymis, seminal vesicle and prostate) was com-parable between the control and treated groups (Table 1).
Changes in sperm concentration and acro-some integrity
In evaluating the effect of long term B. su-perba root extract treatment on hamster sperm quality, the concentration and acrosome status of epididymal sperm were determined. As shown in Fig. 1, the sperm concentration increased signifi-cantly in hamsters treated for 6 months with B. superba root extract in a dose-dependent manner (p<0.05). When the integrity of acrosome on the sperm head was assessed by triple staining (Fig. 2), the percentages of viable sperm with intact acrosome were similar in the control and B. su-perba – treated groups. The percentages of dead sperm with intact acrosome were lower than those of the viable sperm. However, there was no difference between the groups in the percentages of sperm with acrosome, indicating that the treat-ment with B. superba root extract does not affect acrosome integrity of the spermatozoa.
Histological analysis of the testes revealed that a greater abundance of primary spermatocytes and spermatids was observed in the B. superba treated groups than in the control group (Fig. 3a and 3b). No abnormal pathological change was detected in either the control or treated groups.
Change in fertility
In evaluating the fertility of male hamsters after 6 months treatment with B. superba root ex-tract, the percentages of 2-cell embryos collected from the super-ovulated females, after mating with treated males, were compared between the control and treated groups. The percentage of 2-cell embryos was higher at the dose of 1 mg/kg BW as compared with doses of 0.1 and 10 mg/kg BW and the control group (p<0.05) (Table 2).
This study investigated the effects of long term oral treatment of B. superba root extract on sperm quality in the epididymis, and fertility of male hamsters. The results revealed that sperm concentration in the epididymis increased signifi-cantly in B. superba treated-hamsters in a dose-dependent manner. These results are consistent with previous reports of rats treated with crude B. superba powder . Furthermore, histological analysis of testes indicated an increase in number of spermatogonia and spermatozoa in semini-ferous tubules in the B. superba – treated groups. These results indicate that B. superba has a posi-tive effect on the process of spermatogenesis.
The effect of B. superba root extract on sper-matogonia may be due to ß-sitosterol. This phy-tochemical component found in B. superba may be converted to pregnenolone, an important sub-strate of testosterone synthesis in the testes . Testosterone may activate the release of GnRH from the hypothalamus in the treated hamsters. Subsequently, FSH and LH, which are released by the GnRH, may induce spermatogenesis and growth of the spermatozoa during treatment.
Interestingly, the acrosome status of sperma-tozoa in the B. superba – treated groups remained similar to that in the control group. This finding agrees with the effect of B. superba root extract on fertility, when tested by mating with super-ovulated females. The results of this study clear-ly suggest that 6 months of oral treatment with B. superba extract increases spermatogenesis in hamsters without causing adverse effect on the fertility and structural integrity of the reproduc-tive organs.
We thank Ms. Rungthip Posri for her technical help and devotion to this study. This study was supported by the Faculty of Medical Science, Naresuan University, Phitsanu-lok, Thailand. The authors report no conflict of interest.
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