Infertility is a major health problem, and ∼30% of this problem is caused by male factors 1. The percentage of infertility may be high among men in Arabic and Islamic countries because of the traditional beliefs and habits that persist among these men, who may refuse medical consultation for their reproductive problems.
Several conditions can interfere with the process of spermatogenesis and reduce the quality and quantity of sperm. Some diseases and factors such as coronary heart diseases; diabetes mellitus; chronic liver diseases; chronic smoking; insecticide and industrial contaminants; air pollutants; insufficient intake of vitamins; and chronic alcoholism have been reported to exert deleterious effects on spermatogenesis and the production of normal sperm 2. However, some previous studies have reported that intake of antioxidants and vitamins E and C can increase the stability of testicular blood barrier and protect sperm DNA from oxidative stress caused by active free radicals 3.
Zingiber officinale (ZO) (ginger, Family Zingiberaceae) roots are commonly used as a culinary spice and used medicinally for its antioxidant 4 and androgenic properties 5, which have been reported in animal models. Zancan et al. 6 reported that all major active ingredients of ZO roots and leaves such as zingerone, gingerdiol, zingiberene, gingerols, and shogaols have antioxidant activity. In addition, Yang et al. 7 concluded that antioxidants can protect sperm DNA and other important molecules from cell damage induced by oxidation, improve sperm quality, and increase the reproductive efficiency of men. In rats, Khaki et al. 8 reported that ginger has a protective effect against DNA damage induced by H2O2 and may have the potential to enhance healthy sperm parameters. Oral consumption of ginger, dried and powdered, has been shown to result in relief of pain and swelling in patients with rheumatoid arthritis, osteoarthritis, or muscular discomfort 9. Ginger oil has been found to be an inhibitor of cyclooxygenase and lipoxygenase activities 10. Previously, Sharma et al.11 have suggested that ginger oil has anti-inflammatory properties. It has also been reported that dietary ginger protected the tissues from oxidative stress induced by organophosphate pesticide (malathion) in rats 12,13.
In view of the above importance of ginger, it is of interest to study the influence of oral ginger extracts supplementation on sexual dysfunction, fertility, and testis oxidative stress in alcoholic albino rats.
Materials and methods
Dried roots of Z. officinale L. (ginger, Family Zingiberaceae) were obtained from the local market of herbs and medicinal plants. Authentication of the plant was carried out by staff members of the Botany Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt.
As well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee.
White male albino rats (Rattus norvegicus) weighing about 140–180 g were used as experimental animals in the present investigation. They were obtained from the animal house of the Research Institute of Ophthalmology, El-Giza, Egypt. They were kept under observation for about 15 days before the start of the experiment to exclude any intercurrent infection. The chosen animals were housed in plastic cages with good aerated covers at normal atmospheric temperature (25±5°C) as well as 12 h daily normal light cycles. Moreover, they were allowed access to water and supplied daily with a standard diet of known composition and consisting of not less than 20% proteins, 5.5% fibers, 3.5% fats, and 6.5% ash; they were also administered vitamins and mineral mixtures. The rats were divided into four groups and treated as follows:
Group I: Normal control (NC): Six rats received isotonic saline (0.9%) orally through an orogastric tube for equivalent handling.
Group II: Ethanol treatment (Et): Six rats received absolute EtOH orally (10%) in drinking water for 15 days before the experiment 14.
Group III: Ethanol plus ginger aqueous extract (Et+Gt1): Rats were administered orally both EtOH and an aqueous extract of ginger (500 mg/kg) 15 for 4 weeks.
Group IV: Ethanol plus ginger methanolic extract (Et+Gt2): This group of rats received both EtOH and a methanolic extract of ginger (200 mg/kg) 2 for a period of 4 weeks.
Preparation of plant extracts
The methanolic extract was prepared by soaking 100 g of dry ZO roots in 500 ml methyl alcohol 90% with daily shaking for 5 days and kept in a refrigerator.
The watery extract was prepared by soaking 100 g of the dry roots in 500 ml hot distilled water at 40–50°C with daily shaking for 5 days and kept in a refrigerator.
The infusions were filtered and both filtrates were centrifuged at 3000 rpm for 10 min, water was evaporated in a hot-air oven at 50°C, and methanol was evaporated using a rotatory evaporator apparatus that was attached to the vacuum pump.
Induction of alcoholism
Alcoholism was induced experimentally in animals by administration of absolute ethyl alcohol (10%) in drinking water for 15 days before the experiment 14.
The effect of both extracts of ZO roots on the fertility index was estimated using the serial mating technique of the treated males with normal (untreated) females with a regular estrous cycle. The fertility index for each male was calculated as the percentage of number of females that became pregnant in relation to the number of mated females 2.
To quantify mounting behavior, nonestrous female rats were paired with males at the end of the treatment period. Animals were observed for 3 h and their behaviors were scored as described by Lawler 16. Males were placed individually in a separate cage. After 15 min of acclimatization, a nonestrous female was introduced into the arena. The number of mounts was recorded during a 15-min observation period at the start of the first hour. Then, the female was separated for 105 min. Again, the female was introduced and the number of mounts was observed for 15 min as before (third hour). All experiments were conducted from 09:00 to 12:00 h on sunny days (room temperature 27–28°C). A mount was defined operationally as the male assuming the copulatory position, but failing to achieve intromission 17.
At the end of the treatment period, each male was placed in a separate cage. After 1 h, three estrous females were admitted into each cage and they were cohabitated overnight. The stage of the estrous cycle was determined according to the criteria of Ecksterin and Zukerman 17. The vaginal smear of each female rat was examined under a microscope for the presence of sperm. The number of sperm-positive females was recorded in each group.
As in the above experiment, the control and treated males were placed with estrous females for overnight mating. However, in these experiments, each male was cohabitated with three females, with proven fertility. These females were monitored for pregnancy and birth of offspring 17.
Determination of biochemical assays
The testosterone concentration in serum was determined in the Diabetic Endocrine Metabolic Pediatric Unit, Center for Social and Preventive Medicine, New Children Hospital, Faculty of Medicine, Cairo University, according to the method of Andreyko et al. 18, using reagent kits purchased from Biosource Company (Belgium). Serum luteinizing hormone (LH) was determined according to the method of Braunstein et al. 19, using reagent kits purchased from Monobind Inc. (USA). The follicle-stimulating hormone (FSH) concentration in serum was determined according to the method of Odell et al. 20 using reagent kits purchased from Monobind Inc. Testis glutathione (GSH) was determined according to the method of Beutler et al. 21. Testis lipid peroxidation was determined by measuring thiobarbituric acid-reactive substances according to the method of Preuss et al. 22. Glutathione peroxidase (GSH-Px) activity in testis was assayed according to the chemical method of Matkovics et al. 23. Glutathione-S-transferase activity was determined according to Mannervik and Gutenberg 24.
Statistical analysis of the results
The data were analyzed using one-way analysis of variance (PC-STAT, University of Georgia, 1985 25), followed by the fisher's least significant difference test to compare various groups with each other. Results were expressed as mean±SE and values of P value greater than 0.05 were considered nonsignificantly different, whereas those of P values less than 0.05 and less than 0.01 were considered significantly and highly significantly different, respectively.
Autopsy samples were taken from the liver and testes of rats in different groups and fixed in 10% formal saline. The samples were washed in tap water and then serial dilutions of alcohol (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56° in a hot-air oven for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 4 μm by a sledge microtome. The tissue sections obtained were collected on glass slides, deparaffinized, and stained by hematoxylin and eosin stain and then examined using an electric light microscope 26.
Histological examination results showed that there was no histopathological alteration and the normal histological structure of the mature active seminiferous tubules with complete spermatogenic series was observed in group I (Fig. 1). In the ethanol-administered group, histopathological changes such as congestion in the intertubular blood vessels were observed (Fig. 2).
The group administered ginger extracts showed edema, with severe congestion in the blood vessels (Fig. 3).
The alcoholic rats showed a significant decrease in the number of mounts/15 min after 1 and 3 h of mating in comparison with normal rats.
The treatment of alcoholic rats produced a detectable increase in the number of mounts/15 min after 1 and 3 h of mating. However, the effect of aqueous and methanolic extracts of ZO in alcoholic animals was significant after the first and the third hour of mating as compared with the corresponding alcoholic controls. The effect of aqueous extract seemed to be potent (183.33%) after the first hour of mating, whereas with the methanolic extract, it was 133.33%. The effect of aqueous extract also seemed to be more effective (400%) after the third hour (Table 1).
The results obtained showed that oral administration of methanolic extract of ZO roots to male alcoholic rats for 30 consecutive days at doses of 200 mg/kg body weight/day increased the fertility index to 66.67 versus 33.33% in the alcoholic control group and 100% in the normal rats as shown in Table 1. The aqueous extract of ZO, when administered to male alcoholic rats for the same period at doses of 500 mg/kg body weight/day, increased the fertility index to 83.33% (Table 2).
The data of the effect of aqueous and methanolic extracts of ZO on the mating ability and fertility of alcoholic rats are presented in Table 2. The alcoholic rats showed a decrease in mating ability and fertility in comparison with the normal rats. The treatment of these animals with aqueous and methanolic extracts of ZO induced a potential increase in mating ability and fertility (Table 2).
The alcoholic rats showed a significant decrease in the testosterone level, whereas the levels of LH and FSH were increased significantly (P<0.01) in comparison with normal rats (Table 3). The treatment of these animals with aqueous and methanolic extracts of ZO induced a potential increase in the lowered serum testosterone and decreased the elevated LH and FSH levels as compared with the alcoholic animals.
Table 4 shows a significant decrease in the antioxidant levels (GSH-Px and glutathione transferase) in testis of the alcohol group compared with the control one. In contrast, aqueous and methanolic extracts of ginger with alcohol showed significant changes in antioxidant capacities in testis when compared with the control group. Lipid peroxidation level increased highly significantly in the alcohol group compared with the control one; in contrast, groups administered aqueous and methanolic extracts of ginger with alcohol showed a significant decrease as compared with the control one.
Acute alcohol consumption results in testicular injury 27,28, whereas chronic administration results in testicular atrophy and testosterone imbalance in both humans and animals 29. Also, chronic ethanol consumption causes decreased seminiferous tubular diameter and reduced germ cell numbers 29. Both in-vivo and in-vitro studies have indicated that ethanol has direct adverse effects on Leydig cell morphology and function 30. Ethanol-induced impairment of spermatogenesis could reflect a toxic effect of ethanol on Sertoli cell morphology and/or function 30. Creasy 31 reported that one of the most common morphological responses of Sertoli cells to injury is vacuolation and subsequent germ cell degeneration, disorganization, or exfoliation. In our study, the alcohol group showed severe histopathological changes such as congestion in the intertubular blood vessels cells.
After mating of estrous female rats with alcoholic male rats administered either the methanolic or the aqueous extract of ZO roots for 30 consecutive days, the fertility index was high compared with that of the alcoholic nontreated group. This result might be because of the high serum testosterone in the present study. The increases in serum testosterone level, reported in this study, were in agreement with those obtained by Morakino et al. 32, who concluded that the extract of ZO has profertility properties in male rats that might be a product of both its potent antioxidant properties and androgenic activities. Our study supports this suggestion because there was a decrease in the oxidative stress represented by a decrease in lipid peroxidation and increase in the antioxidant enzymes as well as an increase in the serum testosterone level. Moreover, Khaki et al. 8 reported that administration of 100 mg/kg/day of ginger significantly sperm percentage, viability, motility, and serum total testosterone increased. This suggested that ginger may potentially enhance healthy sperm parameters. The author concluded that ZO extract has an androgenic activity in the male rat. Sekiwa et al. 4 isolated two novel glucosides of 6-gingerdiol from fresh ginger (ZO) and investigated their antioxidant activities. The authors concluded that the two glucosides produce strong antioxidant activity by the linoleic acid model system and by their free radicals scavenging ability. Sexual disorders have been reported frequently in chronic alcoholic individuals. The present study on decreased libido and erectile impotence is supported by the studies of Mulligan et al. 33, Rosen 34, and Gumus et al. 35, which showed that high levels of blood alcohol cause reduced sexual stimulation, inability to enjoy orgasms, and retarded ejaculation 36. Furthermore, Whalley 37 reported that 54% of hospitalized alcoholic men and 24% of healthy controls had erectile impotence. Jensen 38 reported that 63% of married alcoholic men and 10% of controls had sexual dysfunction because of lack of sexual desire.
The present study showed increased FSH levels in alcoholic rats. It is the direct toxic effect of alcohol on the testis that leads to decreased seminiferous tubular function. The increase in FSH is because of the absence of testicular feedback regulation at the pituitary level 36. The present results are in agreement with those of Gumus et al. 35, who reported that serum FSH levels are higher in chronic alcoholic individuals. Similar findings have been reported by Van Thiel et al. 39, who found that FSH levels increased in alcohol-fed animals. Because alcohol enters into the testis directly, causing decreases in spermatogenesis and testosterone synthesis, it causes the increased level of LH found in chronic alcoholic individuals 36 as there is no negative feedback by the lowered testosterone level. The results of this study are supported by those of Heinz et al. 40, who found that LH levels were increased in chronic alcoholic individuals. The decreased testosterone and increased LH levels in alcoholic individuals suggest that the major effect of alcohol on serum testosterone level is exerted on the testis at a peripheral site rather than on the hypothalamic–pituitary axis at a central site. This finding is supported by those of Van Thiel et al. 41 and Muthusami and Chinnaswamy 36, who reported that decreased testosterone levels were accompanied by increased LH levels in alcoholic individuals.
In alcoholic individuals, decreased testosterone levels are because of decreased synthesis of testosterone in the testis or increased metabolic clearance of testosterone 36.
The protection system depends mainly on the GSH concentration by the activities of GSH-related enzymes, such as glutathione reductase and GSH-Px 42.
GSH is the principal intracellular antioxidant in most mammalian cells, and participates in the removal of H2O2 and toxic end products of lipid peroxidation known as lipid peroxides 43. It was also reported by Lieber 44 that chronic ethanol consumption depletes the body’s natural antioxidant supply such as GSH; this may explain the decrease in the total antioxidant capacity in the alcohol group compared with the control one. Husain and Somani 45 showed that a single acute dose of ethanol decreased GSH-Px activity significantly in rats. As described in this study, there was a decrease in the testis GSH-Px activity in the alcoholic rat group. These results are also in agreement with those of Ali and Al-Swayeh 46, who reported that ethanol decreased the level of GSH in rats, and suggested that this decrease may be attributed to excess generation of reactive oxygen species (ROS) by ethanol consumption that plays a major role in ethanol-induced oxidative stress 47. Oxidative stress in the cells or tissues refers to enhanced generation of ROS and/or depletion in the antioxidant defense system. ROS generated in the tissues are efficiently scavenged by the enzymatic antioxidant system such as GSH-Px and glutathione reductase as well as nonenzymatic antioxidants such as GSH, vitamin A, C, and E 48.
Fortunately, the alleviation in most investigated biochemical parameters in the alcohol and ginger group presented in the present work could be attributed to the antioxidant properties of ginger. Phenolic compounds such as shogaols and gingerols, zingiberene, zingiberol, curcurmene zingerone, geraniol, and neral in zingiber have antioxidative properties 49. These components may be involved in alleviation of the reduction of the antioxidant capacity and GSH-Px in liver and brain tissues in the alcohol-treated rat groups. This also reflects the decrease in the malonaldehyde level in the alcohol and ginger group when compared with the alcohol group.
The methanolic and aqueous extracts of ZO roots have high safety in rats. The extracts of ginger increase serum testosterone levels and number of mounts, mating, and fertility index of alcoholic control rats in addition to elevation of antioxidant capacities. Therefore, this study recommends that intake of ZO roots as a drink may be useful for alcoholic individuals with sexual impotency. However, further clinical studies are required to assess the safety and efficacy of aqueous and methanolic extracts in humans.
Conflicts of interest
There are no conflicts of interest.
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