International Journal of

Introduction      
Infertility is one of the major health problems in life,  and  approximately  30 %  of  infertilities  are  due to a male factor (1, 2).  Several conditions can interfere with spermatogenesis and reduce sperm quality and production. More factors such as drug treatment, chemotherapy, toxins, air pollutions and insufficient vitamins intake have harmful effects on spermatogenesis and sperm normal production (3). Several studies have reported that antioxidants and vitamin A, B, C, and E in diet can protect sperm DNA from free radicals and increase blood testis barrier stability (4, 5). Nowadays ginger rhizome (Zingiber officinale R., family: Zingiberaceae), is used worldwide as a spice. Both antioxidative (6) and androgenic activity (27) of Z. officinale were reported in animal models. All major active ingredients of Z. officinale, such as Zingerone, Gingerdiol, Zingibrene, gingerols and shogaols, have antioxidant activity (7). Besides, other researches showed that ginger oil has dominative protective effect on DNA damage induced by H₂O₂ and might act as a scavenger of oxygen radical and might be used as an antioxidant (8).
     Antioxidants protect DNA and other important molecules from oxidation and damage, and can improve sperm quality and consequently increase fertility rate in men (9, 10).
     Therefore, the role of nutritional and biochemical factors in reproduction and sub- fertility treatment is very important. The present study was planned to asses the ability of  ginger to promote sperm parameters and modulate follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone concentration, spermatogenesis and oxidative stress. The results obtained will provide further insights into appropriate treatment of male patients by improving spermatogenesis and sperm parameters.
 
Materials and methods
 
Experimental animals
     Adult Wistar albino male rats (n=30) were included in the present study. The rats were 8 weeks old and weighing 250±10g each. They were obtained from animal facility of Pasture Institute of Iran. Male rats were housed in temperature controlled rooms (25°C) with constant humidity (40-70%) and 12h/12h light/ dark cycle prior to experimental protocols. All animals were treated in accordance to the Principles of Laboratory Animal Care. All rats were fed a standard diet and water. The daily intake of animal water was monitored at least one week prior to start of treatments in order to determine the amount of water needed per experimental animal. Thereafter, the rats were randomly divided into control (n=10) and experimental (n=20) groups. The control group just received 4CC distilled water daily. However, the experimental groups split into two groups each included ten rates. (G.1) received 50mg/kg/rat and (G.2) received 100mg/kg/rat of ginger for 20 consequence days. Body weight daily intake of food and water were determined several times per week throughout the study (11).

Surgical procedure
   In twentieth day, the Pentobarbital sodium (40 mg/kg) was administered intra peritoneal for anesthesia, and the peritoneal cavity was opened through a lower transverse abdominal incision. Thereafter testis in control and experimental groups were immediately removed. The weights of testis in each group were registered. The animals were decapitated between 9:00 AM and 11:00 AM, and blood samples were obtained. Blood samples were centrifuged at 4°C for 10 min at 250Xg and the serum obtained was stored at −20°C until assayed.    

Epididymis sperm count, viability and motility
    Sperms from the cauda epididymis were released by cutting into 2 ml of medium (Hams F10) containing 0.5% bovine serum albumin (11).After 5 min incubation at 37°C (with 5% CO2), the cauda epididymis sperm reserves were determined using the standard hemocytometric method and sperm motility was analyzed with microscope (Olympus IX70) at 10 field and reported as mean of motile sperm according to WHO method (12).

Serum FSH, LH total testosterone hormone measurements
Serum concentration of FSH and LH were determined in duplicated samples using radioimmunoassay (RIA). Rat FSH / LH kits obtained from Biocode Company-Belgium, according to the protocol provided with each kit. The sensitivities of hormone detected per assay tube were 0.2ng/ml and 0.14ng/ml for FSH and LH respectively. Serum concentration of total testosterone was measured by using a double antibody RIA kit from immunotech Beckman Coulter Company-USA. The sensitivities of hormone detected per assay tube were 0.025ng/ml (13, 14)

Total antioxidant capacity (TAC) and Malondialdehyde (MDA) concentration measurement in serum
     A TAC detecting kit was obtained from Nanjing Jiancheng Bioengineering Institute-China. According to this method, the antioxidant defense system, which consists of enzymatic and non-enzymatic antioxidants, is able to reduce Fe3+ to Fe2+. TAC was measured by the reaction of phenanthroline and Fe2+ using a spectrophotometer at 520 nm. At 37°C, a TAC unit is defined as the amount of antioxidants required to make absorbance increase 0.01 in 1 mL of serum (15). Free radical damage was determined by  
 Table I. The effect of the 50mg/kg/rat and 100mg/kg/rat ginger on sperm parameters, serum FSH, LH, total Testosterone and testis weight of control and experimental groups in the rats.

 
Result of light microscopic study
     Histopathological study showed the cycle of spermatogenesis was regular in all experimental and control group. However, in all animals exposed to 50mg/kg/rat and 100mg/kg/rat ginger accumulations of sperm, in lumen of seminiferous tubules were seen (Figure 1).


Figure 1. A) Regular seminiferous tubule with normal germinal epithelium morphology, (x640). B) Regular seminiferous tubule with normal germinal epithelium morphology in 50mg/kg/rat of ginger (G.1) group. (x640). C) Regular seminiferous tubule with normal germinal epithelium morphology and sperm presence In lumen (arrow) in 100mg/kg/rat of ginger (G.2) group. (x640).

Discussion
     The main pharmacological actions of ginger and compounds isolated there from include immuno-modulatory, anti-tumorigenic, anti-inflammatory, anti-apoptotic, anti-hyperglycemic, anti-lipidemic and anti-emetic actions. Ginger is a strong anti-oxidant substance and may either mitigate or prevent generation of free radicals. It is considered a safe herbal medicine with only few and insignificant adverse/side effects (17).Oxidants and antioxidants have attracted widespread interest in nutrition research, biology and medicine. It has become clear that constant generation of pro-oxidants, including oxygen free radicals, is an essential attribute of aerobic life (18). A disturbance in the pro-oxidant/antioxidant system has been defined as oxidative stress. Reactive oxygen species (ROS) are very reactive molecules ranked as free radicals owing to the presence of one unpaired electron such as a superoxide ion (O─2 ), nitrogen oxide (NO) and hydroxyl radical (HO─). Even though naturally present in the organism, they are mainly confined to cell compartments and counterbalanced by natural antioxidant molecules, such as glutathione, glutathione peroxidase, superoxide dismutase, vitamin E and vitamin C, acting as free radical scavengers (19,20).
     Ginger extracts have been extensively studied for a broad range of biological activities, especially antioxidant activities (21) found that ginger significantly lowered lipid per oxidation by maintaining the activities of the antioxidant enzymes –super oxide dismutase, catalase and glutathione peroxides in rats. Cellular damage in the semen is the result of an improper balance between ROS generation and scavenging activities. Excessive ROS production that exceeds critical levels can overwhelm all antioxidants defense strategies of spermatozoa and seminal plasma causing oxidative stress (22, 23).
     Therefore, ROS production and total antioxidant capacity (TAC) can be used as a marker of oxidative stress in seminal fluid and is correlated with male infertility. Infertile men with male factor or idiopathic diagnoses had significantly lower ROS-TAC scores than controls (24).
     Besides, Said et al (2005) suggested that abnormal sperm morphology combined with elevated ROS production may serve as a useful indicator of potential damage to sperm DNA. On the other hand, spermatozoa are highly susceptible to damage by excessive concentrations of ROS due to the high content of polyunsaturated fatty acids within their plasma membrane. The lipid peroxidation destroys the structure of lipid matrix in the membranes of spermatozoa, and it is associated with loss of motility and impairment of spermatogenesis (24).
     In the present study, administration of 50mg/kg/rar and 100mg/kg/rat ginger for twenty consecutive days significantly increased sperm motility and viability in both experimental groups as compared with the control group (Table I). These results are supported by the finding of Aitken et al (1995), who reported that the conventional basic semen characteristics other than motility are not obviously influenced by the oxidative state of semen (25). This increase in sperm motility of experimental groups in comparison to control group could be due to the protective effect of ginger rhizoma administration. Besid, these productive effects is reflected by the decrease of malonaldehyde level and increase in total anti oxidants capacity (Table I).
     In according with these results, Hamza et al (2006) have demonstrated that Z. officinale treatment increased the activities of testicular antioxidants enzyme and restore sperm motility of cisplatin-treated rats. Amr et al (26), reported in animal models that Z. officinale have protective effects against cisplatin-induced testicular damage and oxidative stress in rats. Ginger rhizome contains a wide variety of both antioxidative (6) and androgenic activity (27).
     The major active phenolic ingredients isolated from Z. officinale (Zingerone, Gingerdiol, Zingibrene, gingerols and shogaols) have antioxidant activity (7, 27, 28). Others reported that Z. officinale extracts have a potent androgenic activity in male rats (26). In agreement with these reports; the present study showed an increase in the testes weight, serum testosterone levels and accumulations of sperm in the lumen of seminiferous tubules (Figure 1).
     In conclusion, the present study has demonstrated that, ginger possess an antioxidant and androgenic activity in doses of 50 mg/kg/rat and 100mg/kg/rat and have a useful effects on spermatogenesis and sperm parameters in rats.
 
Acknowledgment
     The authors are grateful to the staff at Islamic Azad University Tabriz branch for their help and support and also to Tabriz Porcina herbal shopping center.