The Effects of Methanolic Extracts of Ginger (Zingiber officinale) on Human Sperm Parameters; An in vitro Study

Our understanding of male reproductive function and the importance of male factors in infertility has advanced significantly over the last decade. Infertility is defined as the inability to achieve a pregnancy after one year of unprotected intercourse (Jain et al., 2004). A multi faceted therapeutic approach to improving male fertility involves identifying harmful environmental and occupational risk factors, while correcting underlying nutritional imbalances to encourage optimal sperm production and function (Sheweita et al., 2005). A number of nutritional therapies have been shown to improve sperm counts and motility, including carnitine (Sigman et al., 2006), folic acid and zinc (Wong et al., 2002), selenium (Hawkes and Turek, 2001), vitamin B₁₂ (Boxmeer et al., 2007). Numerous antioxidants have also proven beneficial in treating male infertility such as vitamin C (Sonmez et al., 2005), vitamin E (Keskes-Ammar et al., 2003), glutathione (Meseguer et al., 2004) and coenzyme Q₁₀ (Angelitti et al., 1995). One type of alternative medicine, herbal therapy for medical aliments, recently has become popular despite the lack of scientific experimentation to assess its effectiveness and safety (Ondrizek et al., 1999). Specific botanical medicines have been documented in several studies as having a positive effect on sperm parameters. Chen et al. (1999) found extracts of panax notoginseng were capable of significantly enhancing in vitro sperm motility. When 18 water extracts of Chinese medicinal herbs were evaluated for their effect on sperm motility in vitro, Astragalus membranaceous was the only herb with a significant stimulatory effect (Hong et al., 1992). On the other hand, high concentration extract of some medicinal plants such as saw-palmetto, Echinacea Purpura, ginkgo biloba, st. John`s wort induced inhibitory effects on sperm motility (Ondrizek et al., 1999).

Ginger (Zingiber officinale), a well-known spice, sweet, pungent, heating appetizer has been used in traditional oriental medicines for long time. Its extract and major pungent principles have been shown to exhibit a variety of biological activities (Ghayur and Gilani, 2005; Wei et al., 2005). It has been used to treat a number of medical conditions, including headache, colds and arthritis (Grant and Lutz, 2000). Ginger reduces symptoms in patients with nausea of pregnancy, motion sickness and postoperative nausea and vomiting (Grontved et al., 1988; Phillips et al., 1993). Limited in vitro studies on ginger have shown that water and organic solvent extracts of ginger possess antioxidant active components (Masuda et al., 2004; Reddy and Lokesh, 1992; Krishnakantha and Lokesh, 1993). Ginger significantly decreases lipid preoxidation by maintaining the activities of the antioxidant enzymes (Liu et al., 2003).The main antioxidant active materials in ginger are the gingerols and shogaols and some related phenolic ketone derivatives (Cao et al., 1993). Oxidative stress can induce injuries on the function of cells. Research indicates that oxidative stress can be harmful to sperm survival and fertility and defective sperm function is the most common cause of infertility (Aitken and Baker, 2004). On the basis of the antioxidative effect of ginger, this study was performed to investigate the effect of methanolic extract of ginger on human sperm parameters in vitro.

Semen sampling:Semen samples were obtained from 30 healthy men who referred to IVF center of Babolclinic hospital (located in Babol town, the North of Iran) during 2006. The men who had varicocele, azoospermia or any male factors and age less than 20 years and more than 40 years were excluded from the study.

Sperm preparation and parameters:Semen samples were collected by natural coitus in sterile containers. Semen Fluid Analysis (SFA) was done according to the WHO standards criteria (WHO, 1992). In this procedure fresh sperm specimens were washed using by Ham`s F₁₀ medium and placed in testing tubes. After swim up several smears were prepared from each specimen to record possibly abnormal morphologic pattern. A total of 200 sperms were evaluated per smear in all tests. The sperm count was obtained as million per milliliter. Motility was evaluated by direct examination and morphology by Papanichola staining technique (WHO, 1992). The sperm morphologic assessment at 0 and 8 h of incubation was performed following the method by Kruger et al. (1988). In brief, 5 μL semen were used to make the smears using two morphology slides cleaned thoroughly with 70% ethanol before use. The slides were air-dried at room temperature and fixed for 15 sec.

Experimental groups:The prepared sperm samples were randomly divided into six groups. Different concentrations of methanolic extract of ginger (0.1, 0.2, 0.4 and 0.6%) in sunflower oil as vehicle were prepared. Then 0.9 mL of final sperm in medium was separately mixed with 0.1 mL of vehicle oil and the ginger extracts. Four groups of sperm samples were mixed with different concentrations of ginger extracts. One group of sample was mixed with vehicle and the remaining had no received any treatment. The sperm parameters including motility and grading in 5 steps at 0, 2, 4, 8 and 24 h were evaluated in all study groups. Then smear was provided at 0 and 8 h phases and sperm morphology evaluated by papanichola staining.

Statistical analysis:Sperm motility and grading values are presented as mean±SD. To analysis of within group differences in baseline data repeated measures analysis of variance and for between groups analysis, one way ANOVA with post-hoc Tukey test were done. The difference at 0 and 8 h in sperm morphology was analyzed by Wilcoxon U-test. The different between data with p-value under 0.05 was considered statistical significant.

There was not a significant change in the sperm counts in both of no-treatment and treatment medium with vehicle oil. But the different concentrations of ginger could reduce the count of sperms. The count was significantly reduced with higher concentration of ginger (p<0.05).

The parameter of motility was investigated. No effects were seen with vehicle and no-treatment arms of study. On the other hand, ginger can dose-dependently exert a decreasing effect on sperm motility. On the basis of grading of motility, there were not detected spermatozoa with grade 4 (Table 1).

According to Table 1, as the dose of ginger become increased the level of motility will be decreased. This reduction profile of the sperm motility was significantly time dependent (p<0.001).

In this investigation all sperms which were exposed to the different concentration of ginger were classified under grading levels of motility. With except of grade 4, other grades of motility were presented in Table 2-4. All sperms were exposed to the ginger concentrations showed a reduction in the motility grading. Long duration of exposure of the sperms to different concentrations of ginger can resulted in formation low grade motility of sperms. The grade 1 of motility was seen after exposure to ginger concentration. But this reduction in grade 1 of motility with treatment of 0.6% concentration of ginger was seen higher than other concentrations of ginger. On the other hand, the trend of reduction of motility from grade 3 and 2 into grade 1 with 0.6% concentration was quicker than other concentration. After time consuming of exposure to high concentration of ginger all sperms became immotile.

The mean percentage of sperm frequency in Grade 1 motility increased after exposure to different concentrations of ginger. This increase at hour 2, 4 and 8 but no 24 were seen in concentrations higher than 0.1 (p<0.0001).

Table 1:	The mean±SD of percent sperm motility in various concentrations of methanolic extract of ginger by times

Table 2:	The mean±SD percentage of sperm frequency in grade 1 motility in different concentrations of methanolic extract of ginger in various time

Table 3:	The mean±SD percentage of sperm frequency in grade 2 motility in different concentrations of methanolic extract of ginger in various times

Table 4:	The mean±SD percentage of sperm frequency in grade 3 motility in different concentrations of methanolic extract of ginger in various times

This position was not seen in sperm of grade 2. This sperms slightly shifted into grade 1 and after this step all the sperms will be significantly immotile.

The frequency of grade 2 of sperm motility dose dependently was decreased in the present of various ginger concentrations.

With the except of 0.1% concentration of ginger, the motility of grade 3 of sperms was more rapidly reduced to zero in the exposure to higher concentrations.

AS can be seen in Table 4, a dose and time- dependent reduction were observed in percentage of sperm frequency in grade 3 of motility (p<0.0001).

Table 5:	The mean±SD percentage of sperm frequency with normal morphology in different concentrations of methanolic extract of ginger

A significant decrease in the number of spermatozoa with normal morphology is observed after exposure to all concentrations of ginger extracts (Table 5).

In this study, the effect of different doses of methanolic ginger extracts were investigated.

Several in vitro studies similar to present study investigated the effects of herbal medicine on sperm motility. Amano et al. (1996) found that the motility of sperm in 10 and 100 μg mL^-1 of Hochu-ekki-to (Astragalus), a Chinese herbal medicine, for 1-6 h was significantly than that of the control groups.

Ginger extracts have been extensively studied for a broad range of biological activities, especially antioxidant activities. Ahmed et al. (2000) 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. The results indicate that ginger is comparatively as effective as ascorbic acid as an antioxidant. In the other hand, we know that spermatozoa membranes are rich in poly-unsaturated fatty acids and are sensitive to oxygen induced damage mediated by lipid preoxidation. The cellular damage in the semen is a result of an improper balance between oxygen free radicals generation and scavenging activities. Therefore numerous antioxidants such as vitamin C, vitamin E, glutathione and coenzyme Q₁₀ have proven beneficial effects in treating male infertility (Sheweita et al., 2005). But a number of certain drugs such as diltiazem (Wood et al., 2003), colchicines (Haimov-Kochman and Ben-Chetrit, 1998), lidocaine (Moudgil et al., 2002) and chemotherapeutic agents (Longhi et al., 2003) inhibited sperm motility in vitro.

Also smoking and heavy use of alcohol can decrease semen quality (Martini et al., 2004). To evaluate protective effect of ginger against cisplatin-induced reproductive toxicity, ethanolic extract of Zingiber officinale (1 g/kg/day) were given P.O. to male albino rats for 26 days. Result showed that ginger treatment increased the activities of testicular antioxidant enzymes and restored sperm motility of cisplatin-treated rats (Amin and Hamza, 2006), which was in contrary with present results. Also the aqueous extract of Z. officinale significantly increased weight of the testis, the serum testosterone level and epididymal a-glucosidase activity in vivo (Kamtchouing et al., 2002).

Despite the antioxidants effect of ginger which is mentioned above, all concentrations of ginger in this study had time-dependent damaging effects on sperm motility and morphology. These effects could be due to administration of high doses of ginger in this investigation. A clinical trial study showed that high concentrations of saw-palmetto, echinacae, or ginkgo inhibited motility at 24 and 48 h (Ondrizek et al., 1999).

This result is consistent to present study that ginger extract in this investigation can reduce the sperm motility.

In this study, after 8 h the percentage of grade 1 sperms in all concentrations except of 0.6% were increased. This temporary increase can be arise from simultaneous decline in the number of grade 2 sperms that is some of grade 2 sperms were imported in grade 1 phase.

In conclusion, these data indicate that some exhibits a lower percentage of motility and grading when methanolic ginger is added to semen fluid. According to the results it would be concluded that ginger can induce the decreasing effects on sperm parameters. In fact in our study, ginger extracts had adverse effect on sperm motility and morphology in vitro which is dose and time dependent.