EFFECT OF Nigella sativa OIL TREATMENT ON THE SEX ORGANS AND SPERM CHARACTORS IN RATS EXPOSED TO HYDROGEN PEROXIDE

The effect of Nigella sativa oil (0.8ml/kg B.W) on testis, accessory sex glands and epididymal sperm characters was studied in mature male albino rats treated with hydrogen peroxide H2O2(0.5% in drinking water) for 30 days orally. The results showed thatH2O2 treatment caused a significant decrease in the percentage of live/dead sperms 13.66% associated with a significant increase in the percentage of and morphologically abnormal sperms 39.5%.While a significant increase in the percentage of live/dead sperms in both Nigella sativa oil treated group and Nigella sativa oil joined to H2O2 (85%), (72.16%) , respectively accompanied with a decrease in percentage of morphologically abnormal sperms compared with H2O2 treated group. It is concluded that Nigella sativa oil possess an antioxidative actions to counter act the impairment in the epididymal sperm characters caused by H2O2 treatment. INTRODUCTION Oxidative stress is a condition associated with an increased rate of cellular damage induced by oxygen derived species, a highly reactive oxidizing agents, belonging to the class of free radicals (Sikka etal., 1995). The most common reactive oxygen species that have potential implications in reproductive biology include superoxide (O2) anion, hydrogen peroxide (H2O2), peroxyl (ROO) radicals and the very reactive hydroxyl (OH') radicals. The nitrogen–derived free radical nitric oxide (NO') and peroxy nitrite anion (ONOO') also appear to play a significant role in reproduction.(Sikka, 1996). Excessive reactive oxygen species generation can overwhelm the protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membrane. Additionally changes in DNA can be induced (Sanocka and Kurpisz, 2004). Mammalian sperm cells have high content of specific lipidic composition of polyunsaturated fatty acids, plasmalogenes and sphingomyelins (Aitken etal., 1989).This unusual structure of sperm membrane is responsible for its flexibility and the functional ability of sperm cells, However, spermatozoa's lipids, are the main substrates for peroxidation, that may provoke severe functional disorder of sperm (Aitken etal.,1989). On the other hand, antioxidants, in general, are compounds and reactions which dispose, scavenge and suppress the formation of ROS or oppose their reactions (Sikka, 1996). Recently, dietetical antioxidants and their roles in free radical removal and prevention of tissue, cellular lesion have garnered attention especially for their usefulness in reproduction and management of infertility.Nigella sativa oil have known for their antioxidant properties in ameliorating inflammatory received 25/5/2005 accepted 28/2/2006 Mesopotamia J. of Agric (ISSN 1815-316 X) Vol. ( 34 ) NO ( 1 ) 2006 disease (EL-Dakhakhny etal., 2002).In addition, Mahmoud etal. (2002) pointed to the role of Nigella sativa oil against mice liver damage caused by Schistosoma mansoni infection by improving the immunological host system with its antioxidant effect. On the other hand, Black cumin seed oil enhances potency and have aphrodesiac action (Laver, 1984).The present study was designed to investigate the anti oxidative effect of Nigella sativa oil on testis, accessory sex glands, epididymal sperm characteristics in adult male rats exposed to oxidative stress induced by hydrogen peroxide. MATERIALS AND METHODS 1.Extraction of oil from Nigella sativa seeds:The Nigella sativa seeds were purchased from local herb grocery in Mosul city.The seeds were ground in porcelain mortar.one Kg of the powdered seeds with appropriate amount of ethanol were heated to(80-90C ̊)in the extraction apparatus (soxhelet) (Maynard, 1970). Heating and extraction continued until uncoloured solvent was collected. This distillate was extracted by steam distillation until clear water was obtained. Moisture was removed by anhydrous sodium sulphate and the resultant extract was evaporated using water bath (40C ̊), this led to obtain the oil which was kept in a dark flask (Harvey and John,1998). The percentage of oil extracted was calculated by the following equation: Percentage of oil = ×100 The percentage of black seed oil was 12.7 %. 2. Experimental design: Twenty four male albino rats of 90-100 days of age were used, they were housed in polypropylene cages under controlled conditions of temperature(24-26C ̊)and natural lighting (10 hours light/14 hours dark)food and water were supplied ad libitum. The animals randomly divided into four groups (6 rats/group). The first group received normal saline and served as control.The second group received 0.5%H2O2 (Al shaheed factory-Iraq) in drinking water(Matkovics,1977).The third group received Nigella sativa oil (0.8ml/kg B.W) orally by gavage needle.The fourth group received 0.5%H2O2 in drinking water and Nigella sativa oil (0.8ml/kg B.W)orally by gavage needle. At the end of thirty days experimental period, the animals were scarificed by the ether administration and their weights were recorded.The abdominal cavity was opened, Testis, Epididymis (head, body,tail),prostate and seminal vesicle were immediately removed, trimmed, blotted in folds of filter paper and weight.Total epididymal head sperm count in 1ml was determined according to Sakamoto and Hashimoto oil weight


MATERIALS AND METHODS
1.Extraction of oil from Nigella sativa seeds:The Nigella sativa seeds were purchased from local herb grocery in Mosul city.The seeds were ground in porcelain mortar.oneKg of the powdered seeds with appropriate amount of ethanol were heated to(80-90C˚)in the extraction apparatus (soxhelet) (Maynard, 1970).Heating and extraction continued until uncoloured solvent was collected.This distillate was extracted by steam distillation until clear water was obtained.Moisture was removed by anhydrous sodium sulphate and the resultant extract was evaporated using water bath (40C˚), this led to obtain the oil which was kept in a dark flask (Harvey and John,1998).The percentage of oil extracted was calculated by the following equation: Percentage of oil = ×100 The percentage of black seed oil was 12.7 %.

Experimental design:
Twenty four male albino rats of 90-100 days of age were used, they were housed in polypropylene cages under controlled conditions of temperature(24-26C˚)and natural lighting (10 hours light/14 hours dark)food and water were supplied ad libitum.The animals randomly divided into four groups (6 rats/group).The first group received normal saline and served as control.The second group received 0.5%H 2 O 2 (Al shaheed factory-Iraq) in drinking water (Matkovics,1977).(1986).The right epididymal head was dissected into small parts in petri dish with the addition of 9.8 ml neutral formalin buffer and 0.1ml of 5% eosin stain.Sperm count was done using hemocytometeric technique.The percentage of live, dead and morphologically abnormal sperms were counted using eosin-nigrosin stained smears.Data were analyzed statistically using oneway analysis of variance.Group differences were determined using Duncan test (Steel and Torrie,1960).The level of significance was at p < 0.05.

RESULTS AND DISCUSSION
Treatment with H 2 O 2 (0.5%) in drinking water for 30 days caused a significant (p<0.05)decrease in epididymal head weight (Table1), the percentage of live/dead sperms with a significant (p<0.05)increase in the percentage of morphologically abnormal sperms (Table2) as compared with the control group.Oral administration of Nigella sativa oil (0.8ml/kg B.W) for 30 days showed a significant (p<0.05)decrease in the percentage of morphologically abnormal sperms with an increase in the percentage of live/dead sperms (Table2) as compared with the H 2 O 2 -treated groups.On the other hand, the group treated with 0.5% H 2 O 2 and 0.8 ml/kg B.W. Nigella sativa oil revealed a significant (p<0.05)increase in weights of the testis, epididymis (head and body) and seminal vesicle (Table1),the percentage of live/dead sperms with an associated a significant (p<0.05)decrease in the percentage of morphologically abnormal sperms (Table2) when compared with the group treated with H 2 O 2 .The results of the present study demonstrated that H 2 O 2 -induced oxidative stress resulted in a decrease in epididymal head weight accompanied by disturbance of sperm characters in rats.The H 2 O 2 -induced oxidative stress in the present study may lead to the production of high amounts of reactive oxygen species.One mechanism suggests that H 2 O 2 administered orally probably increases oxygen production in the stomach which reaches the blood and increases oxygen tension in tissues causing increased production of reactive oxygen intermediates (Loven and Obrelery, 1985).These intermediates may be responsible for the decrease in the percentage of live sperms and increased percentage of dead sperms and morphologically abnormal sperms.In the male reproductive tract, oxidative stress may create aground for sperm deterioration and transitory or persistent infertility (Kurpisz, 2004).The results of the present study seem to agree with those obtained by Jassem and Yousif (2004) in rats and Aziz (2000) in mice.Mazilli et al. (1994) found a positive correlation between O 2 levels and sperm abnormalities and the increase in reactive oxygen species, including, Superoxide anion, caused increase in the percentage of dead, morphologically abnormalities with a decrease in percentage of live sperms in human.Peroxidation of poly unsaturated fatty acids has implicated in a wide variety of pathological conditions, infertility amongst them agree with suggestion by Atiken et al.(1989) that, low(physiological) levels of lipid peroxidation reflect the influence of reactive oxygen species on sperm metabolism enhancing the ability of spermatozoa to interact with zona pellucida while higher, pathological lipid peroxidation of sperm membranes can be unbalanced oxidative stress.It has been known for along time that oxygen metabolism can be deterimental to the cells and tissue and ROS release has effects on male infertility.Sanocka et al.(2003)reported that the unpaired electrons make the external orbit vulnerable and imitate the effect of ionizing radiation within the tissue affecting the lipids, proteins and DNA.Lipid components located in the sperm membranes are involved in regulation of sperm maturation, spermatogenesis, capacitation, acrosome reaction and eventually in membrane fusion and so peroxidation of sperm lipids may disturb sperm functions which in extreme cases even completely inhibit spermatogenesis (Sanocka and Kurpisz,2004).Interestingly, antioxidants seem to offer new ways of conservative therapy that is currently used in a variety of diseases such as infertility.Sikka et al. (1995) mentioned that there is a variety of biological and chemical antioxidants that attack ROS present in seminal fluid and, in turn help spermatozoa to comabat oxidative insult maintaining sperm motility and function.On the other hand, Gavella et al. (1996) reported that infertile men had lower antioxidant status level than fertile men, especially those with poor sperm motility.Oxidative stress revealed improper balance between ROS generation and scavenging activities.The scavenging potential in gonads and seminal fluid is normally maintained by adequate level of antioxidants superoxide dismutase (SOD), catalase and probably glutathione (GSH), peroxidase and reductase (Sikka et al., 1995).The results of the present study demonstrate the beneficial effect of the treatment with Nigella sativa oil as manifested by the(85%) increase in the percentage of live/dead sperms, and (0.66%)decrease in the percentage of morphologically abnormal sperms reflecting its antioxidants effect that counteract the H 2 O 2 effect on sperms, also black seed oil enhances potency.It has a powerfulally in restoring sexual performance by renew virility by enhancing the production of body fluids, male hormones and rejuvenating blood circulation.Nigella sativa oil constituents possessed avariable antioxidant activities.It inhibited the non enzymatic peroxidation in ox brain phospholipidliposomes (Houghton et al.,1995).Nearly similar results were observed by Rady et al. (1997)  have respectable radical scavenging properties, which may give explain the increase in the percentage of live/dead sperms with decrease in the morphologically abnormal sperms by Nigella sativa oil treatment that counteract the H 2 O 2 effect in the present study.It is concluded from this study.That Nigella sativa oil have a protective and antioxidant effect on testicular, accessory sex gland functions in animals exposed to oxidative stress induced by hydrogen peroxide and whether the anti-oxidative action induced by Nigella sativa oil as a results of a direct action or a results of indirect effects, e.g including antioxidative enzymes cascade, is unknown and should be subjected for further investigation.

Table ( 1
): Effect of 30 days oral administration of hydrogen peroxide H 2 O 2 (0.5% in drinking water) and Nigella sativa oil (0.8ml/ kg B.W) on weights of (body, testis , epididymis (head, body tail)and male accessory sex glands in rats.Numbers of animals 6 rats/group.Values are expressed as mean ± SE.Values with different letters are significantly different at P< 0.05 .

Table ( 2
): Effect of 30 days oral administration of hydrogen peroxide (H 2 O 2 )(0.5% in drinking water) and Nigella sativa oil(0.8ml/kgB.W) on epididymal sperm characters in rats.Numbers of animals 6 rats/group.Values are expressed as mean ± SE.Values with different letters are significantly different at P< 0.05.