Introduction. Material and Methods

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1 المجلد/ 5 العدد/ مجلة القادسية لعلوم الطب البيطري The level of reactive oxygen species(ros) in fresh and aged Asthenospermic, Leukocytospermic and Normospermic semen specimens F. N.A.AL-Hady Coll. of Sci.Unive.of Babylon Abstract This study was designed to determine the malondialdehyde (MDA)concentration as a sign of Oxidative Stress (OS), and its effect on sperm function parameters ( sperm motility percent, grade activity, and sperm viability percent) of normospermic (control), asthenospermic and leukocytospermic semen specimens after different incubation periods( half hour, two hours, and four hours).it was showed that MDA level in asthenospermic specimens was significantly increase(p<0.05) when compared to normospermia and leukocytospermia after incubation for half hour and two hours. While both asthenospermic and leukocytospermic semen samples had significant increase(p<0.05) of MDA level compared to normospermia after four hours of incubation.the effect of incubation period on MDA level and sperm function parameters revealed that increasing incubation period caused significant decrease (P<0.05) in sperm motility percent, grade activity, and sperm viability percent in asthenospermic and leukocytospermic semen samples after two hours and four hours of incubation, which accompained with in insignificant increase in MDA level. The spermatozoa produce small amounts of reactive oxygen species (ROS) in specific physiological states which are considered an important to perform capacitation, acrosome reaction and fertilization (Griveau and Le Lannou, 1997). While a large amount of ROS was produced by immature spermatozoa and leukocytes caused an impairment of normal spermatozoa as a results of increasing of lipid peroxidation (Agarwal et al., 2003).The high molecular weight molecules present in seminal fluid which are called antioxidants or scavengers system, provide protection against ROS effects (Pasqualotto et al., 2000).The OS was affected by several factors mainly in leukocytospermic patients. The presence of Thirty seminal fluid specimens were collected from fertile men, asthenospermic and leukocytospermic patients (ten specimens for each group).the seminal fluid specimens were collected by masturbation after 3-5 days of abstinence. The specimens were incubated in 37C for allow to liquefaction.seminal fluid analysis and MDA concentration were performed after half hour, two hours and four hours of Introduction Material and Methods 22 leukocytes mainly granulocytes caused increase ROS and then OS increasing (Novotny et al.,2003). In addition to this the OS correlated with varicocele. A study revealed that ROS level increased with late stage of varicocele(agarwal,2004).another study showed that is a relationship between smoking and increasing of ROS compared to no smoking infertile men(ramadan et al., 2003).The aims of the present work were to: -Usage of MDA as a probe for ROS level in asthenospermic and leukocytospermic patients. -Checking the semen incubation period effects on ROS level and sperm function test in asthenospermic, leukocytospermic and fertile men. incubation. The studied sperm parameters include: sperm motility percent, grade activity, and sperm viability percent (Mortimer et al.,1988; Hintig,1989; WHO, 1992).The estimation of seminal plasma MDA concentration was performed according following steps(muslih et al., 2003): 1- Manipulation 150µL of seminal plasma into dry and sterile glass test tube and add

2 1ml of 17.5% Trichloroacetic acid (TCA) 4- Centrifugation the mixture for 15 and 0.6% of Thibarbituric acid ( TBA ). minutes at 2000 rpm to separate the 2- Placement the test tube in boiled water supernatant. for 15 minutes and then cooling it. 5-Reading the absorption of supernatant by 3- Addition 1ml of 70% TCA and left the using spectrophotometer at 532nm. The mixture for 20 minutes in room MDA concentration evaluated due to temperature. following equation: A 532 MDA conc.(mmol/l)= D A: Absorbance at 532 ; L: Light bath=1cm; E : Extinction coefficient= M cm D: Dilution factor=1ml / 0.15ml = 6.7 A significant increase (P<0.05) of MDA in asthenospermic semen samples compared to normospermic semen samples. While there is insignificant differences (P>0.05) between the leukocytospermic semen samples and both of normospermic and asthenospermic samples. The Results L E The results were analyzed by using analysis of variance(anova)and LSD to indicate the significancy( Sorli,1995). asthenospermic and leukocytospermic semen samples increased significantly (P<0.05)of MDA concentration compared to normospermic semen samples,while there is insignificant differences (P>0.05) between asthenospermia and leukocytespermia after four hour of incubation. Table(1) Malondialdehyde (MDA) concentration in normospermic, asthenospermic and leukocytospermic semen samples after incubation for half hour, two hours and four hours. Groups Incubation Normospermia Asthenospermia Leukocytospermia Period Mean±SE Mean±SE Mean±SE Half hour 5.32±0.57 a 7.99±0.95 b 7.12±1.02 a b Two hours 5.62±0.59 a 8.62±0.94 b 7.76±1.06 a b Four hours 6.05±0.59 a 9.18±0.94 b 8.54±0.92 b No. Of semen specimens 10 for each group. Different letters mean significant differences(p<0.05). The effect of incubation periods on MDA concentration and sperm parameters, showed insignificant increase (P>0.05) of MDA concentration in asthenospermic, leukocytospermic and normospermic semen samples after two and four hours compared to standard incubation period (half hour) (Tables 2,3,and 4).The studied sperm parameters were significantly different between the different incubation periods in all groups. In normospermic semen samples a significant decrease (P<0.05) was noticed in sperm motility percent and grade activity after four hours of incubation compared to incubation for half hour. While there are insignificant differences (P>0.05) between two hours and four hours, two hours and half hour of incubation.sperm viability percent was significantly decreased (P<0.05) after four hour of incubation compared to half hour and two hours of incubation, while there is no significant difference (P>0.05) between two hours and half hour of incubation ( Table-2-).The sperm motility percent, grade activity and sperm viability percent in both asthenospermic and leukocytespermic semen specimens were significantly decreased (P<0.05) as the incubation periods increased (Tables 3 & 4). 24

3 Table(2)Semen MDA concentration and sperm parameters of Normospermic specimens after half hour,two hours and four hours of incubation Parameters Incubation period (Mean± SE) Half hour Two hours Four hours MDA a a a concentration(µmol/l) 5.32 ± ± ± 0.59 sperm motility percent 65.80±4.59 a 54.10±4.90ac 41.20±4.96 bc grade activity 3.25±0.5 a 2.65 ± 0.28ac 1.95 ± 0.25 bc sperm viability percent ± 2.73 a 82.70± 5.21 a ±5.35 b No. Of specimens:10 for each group Different letters mean significant differences(p<0.05) Table(3)Semen MDA concentration and sperm parameters of asthenospermic specimens after half hour, two hours and four hours of incubation Parameters Incubation period (Mean± SE) Half hour Two hours Four hours MDA concentration(µmol/l) 7.98± 0.96 a 8.62 ± 0.94 a 9.24±0.94 a sperm motility percent ±2.57 a 23.50±2.72 b 9.80±2.30 c grade activity 1.90± 0.16 a 1.25±0.20 b 0.65±0.02 c sperm viability percent 64.40±2.70 a 48.10±3.78 b 32.70±2.98 c No. Of specimens:10 for each group. Different letters mean significant differences(p<0.05). Table(4)Semen MDA concentration and sperm parameters of Leukocytospermic specimens after half hour, two hours and four hours of incubation Parameters Incubation period (Mean± SE) Half hour Two hours Four hours MDA concentration(µmol/l) 7.20±1.02 a 7.67±1.06 a 8.24±.098 a sperm motility percent 58.60±3.75 a 44.60±4.24 b 30.80±4.12 c grade activity 2.60±0.15 a 2.10±0.16 b 1.43±0.15 c sperm viability percent 85.30±2.85 a 70.40±4.24 b 56.50±4.75 c No. Of specimens:10 for each group. Different letters mean significant differences(p<0.05). Sperms may generate ROS in two ways: (1) the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system at the level of the sperm plasma membrane (Aitken et al.,1992), and(2): the NADHdependent oxido reductase (diphorase) at the level of mitochondria(gavella and Lipova,1992).The mitochondria system is the main source of ROS in spermatozoa from infertile men (Plante et al.,1994).it was well known that the sperm motility depend mainly on mitochondria system to support the energy for motility, this function may be impaired in Discussion 22 asthenospermic patients due to high ROS production from mitochondria system. Gomes et al.(1998) had indicated that the level of ROS production by pure sperm populations were negatively correlated with quality of the sperms in the original semen.the results of this study showed a significant increase of ROS in asthenospermic semen samples compared to those from normospermia after incubation for half hour, two hours and four hours. Most causes of asthenospermia are unexplained, so that and due to the results of present study, the level of ROS

4 المجلد/ 5 العدد/ مجلة القادسية لعلوم الطب البيطري in asthenospermic samples may be considered as one of those of unexplained causes,because all or most infertility laboratories routinely they not performed the estimation of ROS level. According to these results of present study, it was recommended to evaluate the ROS level for treating asthenospermic patients by exogenous antioxidants like vitamin C and vitamin E.The pathological level of ROS in seminal plasma from infertile men are significantly more than those from fertile men (Zini et al.,1995).the leukocytes are present throughout the male reproductive tract and are found in almost every human ejaculate (Tomlinson et al., 1992). The WHO define leukocytospermia as the presence of peroxidase- positive leukocytes in concentration of >1million/ml of semen (WHO,1999). Peroxidase- positive leukocytes include polymorphonuclear leukocytes, which represent 50% to 60% of all seminal leukocytes and macrophages, which represent another 20% to 30% (Thomas et al.,1997).in the present study, the MDA concentration in leukocytospermic semen samples was insignificantly more than those in normospermic semen samples. Peroxidase-positive leukocytes were found to be the majore source of high ROS production in semen (Rajasekaran et al.,1995; Shekarriz et al.,1995).the results dealing with the effect of incubation periods on Malonaldehyde (MDA) concentration for normospermic, asthenospermic and leukocytospermic semen specimens showed insignificant increase in MDA concentration with increasing of incubation period. These results may be refer to contain the seminal plasma an antioxidants which are act as arrest of ROS action and then reducing lipid peroxidation such as MDA level. The seminal fluid contain several enzymatic antioxidants like superoxide dismutase (SOD), Catalase and Glutathion Peroxidase (GPX), in addition to non-enzymatic antioxidants like vitamin C,vitamin E, carnitin, glutathion and pyruvate (Saleh and Agarwal 2002). The results of our study agree with Kovalski et al.,(1992) 25 study, which are revealed that seminal plasma reduced ROS level in seminal fluid.the effects the incubation period on sperm parameters in normospermic semen specimens showed significant decrease in sperm motility percent, grade activity and sperm viability percent after incubation for four hours compared to half hour and two hours of incubation. Also there was no significant difference between half hour and two hours in above sperm parameters, this may be refered to normal spermatozoal function which resistance the denaturation of seminal components due to aging of semen specimens.the incubation periods of two hours and four hours of infertile semen specimens caused significant decrease in sperm motility percent, grade activity and sperm viability percent, and these reduction increase with increasing of incubation period. This results was in agreement with the study of Engel et al.,(1999) study, which revealed that forward progressive motility was reduced with increasing the age of semen sample. Other study showed that sperm motility index and sperm viability percent of asthenospermic semen samples were significantly decrease after two hours and three hours of incubation, while there are a significant increase of total semen protein with aging of asthenospermic semen sample(aljeboory,2004).the reduction of sperm function parameters with aging of the samples may be refer to the reduction of glucose and fructose concentration in seminal plasma. These sugars act as main source of energy required for sperm function, because the sperm motility and viability depend on fructose and other nutritional substances present in seminal plasma which are consumped during incubation. The sperms not posses nutritional storage except lipids, so that it tend to maintain its ATP formed by peripheral mitochondria in midpiec to perform glycolysis and cell respiration, and the metabolic rate was agreement with sperm motility(page et al.,1981).on the other hand the reduction of sperm function parameters may be referred to continuous exposure of spermatozoa to ROS inside the

5 sperm. Aitken and Fisher(1994) were then inhibit the action of membrane reported that ROS production inside the binding enzymes like Ca+2/Mg+2-ATPase sperm was associated with sperm motility maintaining. Calcium level in the cells decreasing and reduce sperm-oocyte which are necessary for motility and fusion. Also sperm cytoplasm volume activity of the sperms(de Lamirand and should be reduced and then contain less Ganong,1992; Irvine,1996; Wang et amount of antioxidants compared to al.,2003).it was concluded that the ROS seminal plasma which is contain efficient level from infertile semen more than in antioxidants that can arrest ROS action( de fertile semen samples. ROS was increased Laminarde and Ganong,1995; Agarwal et with incubation periods increasing, and al., 2003).The ROS caused losing of exposure to ROS for long period caused energy inside the cells and decrease the impaired of sperm function parameters. mitochondrial membrane potential (MMP), References Agarwal,A.(2003). Significance of oxidative stress and sperm chromatin damage in male infertility. In:Male fertility and lipid metabolism,(eds.s.devriese and A.Christophe), AOCS Press, Champaign,IL.Chapter13:pp.157. Agarwal,A. (2004). Increased seminal reactive oxygen species levels in patients with varicocele grade but not with testis size. Fertil. Steril., 82: Agarwal,A.;Saleh,R.A.and Bedaiwy, M.A. (2003).Role of reactive oxygen species in the path physiology of human reproduction. Fertil.Steril., 79: Aitken,R.J.;Buckingham,D.W.andWest,K. M. (1992). Reactive oxygen species and human spermatozoa: analysis of the cellular mechanisms involved in Luminaland lucigenin-dependent chemiluminescence. J. Cell Physiol., 151: Aitken,R.J.and Fisher,H.(1999).Reactive oxygen species generation and human spermatozoa: the balance of benefit and risk. Bioassay, 16: Aljeboory,A.H.(2004). The effect of androgens on sperm activation of aged semen in vitro. M.Sc. thesis, Babylon University. de Lamirands,E.and Ganong,C.(1992). Reactive oxygen species and human spermatozoa.1- Effect on motility of intact spermatozoa and 22 on spermatozoa and on sperm axonema,and 2- Depletion of adenosine triphosphate plays an important role in the sperm motility. J. Androl.,13: de Laminarde, E. and Ganong,C. (1995).Impact of reactive oxygen species on spermatozoa: a balancing act between beneficial and detrimental effects.hum. Reprod., 10: Engel,S.;Schreiner,I.and Petzold,R. (1999). Lipid peroxidation in human spermatozoa and maintains of progression sperm motility. Andorologia,31: Gavella,M. and Lipovac,V.(1992).NADHdependent oxidoreductase (diaphorase) activity and isozyme pattern of sperm in infertile men. Arch.Androl.,28: Gomez,E.;Irvine,D.S. and Aitken,R.J. (1998). Evaluation of a spectrophotometric assay for measurement of malondialdehyde and 4-hydroxy Alkenals in human spermatozoa: relationships with semen quality and sperm Function. Int.J. Androl.,21: Griveau,J.F. and lelannou,d. (1997). Reactive oxygen species and human spermatozoa: physiology and pathology. Int.J.of Androl., 20: Hinting, A.(1989).Method of semen analysis in: Assessment of human sperm fertilizing ability. Ph.D. thesis, Michigan University.

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7 seminal leukocytes. Hum. World Health Organization. (1999). Reprod., 7: Laboratory manual for the Wang,X.;Sharma,R.K.;Ggpta,A.;George,V examination of human semen and.;thomas, A.J.; Falcon, T. and sperm-cervical mucus interaction. Agarwal, A.(2003).Alteration in 4 th ed. Newyork :Cambridge mitochondria membrane potential University press. and oxidative stress in infertile Zini, A.;deLamirand,E. and Ganong,C. men: a prospective observational (1995). Reactive oxygen species study. Fertil.steril.,80: in the semen of infertile patients: World Health Organization(1992). levels of superoxide dismutase laboratory manual for the and catalase-like activities in examination of human semen and seminal plasma. Fertil.Steril., semen-cervical mucus interaction. 64: rd ed. Cambridge University press, Cambridge. مستوى األنواع األوكسج ن ة الفعالة ف ع نات السائل المنوي الطازجة والمعمرة لمرضى وهن النطف واب ضاض المن والرجال الخص ب ن فارس ناجي عبود الهادي كلية العلوم/جامعة بابل الخالصة صمممه هملد الدراسمة لتقمدير تركيما المالونمداي الديهايمد كعةممة للجهمد التأكسمدي وتأثيرهما علما معمالم النطم والتمي شمله: النسبة المئوية للنط المتحركة ودرجة نشاط النط والنسبة المئوية لعيوشية النط لعيناه المني الطبيعية ووهن النطمممم وابي مممماض المنممممي ل تممممراه الح ممممن الم تل ة نصمممم سمممماعة وسمممماعتين وظربمممم سمممماعاه لوح ه ايمممماد معنوية 0.05>P في تركيا المالونداي الديهايد لعينماه وهمن النطم مقارنمة مم كمل ممن عينماه منمي الرجمال ال صميبين وعيناه المني المصابة بابي ماض المنمي ل تمراه الح من نصم سماعة وسماعتين بينمما ااداد تركيما المالونمداي الديهايمد معنويا 0.05>P في عيناه مني مر ا وهن النط وابي اض المني بعد الح ن ظرب سماعاه ظمما فيمما ي مث تمأثير فتراه الح ن علا تركيا المالونداي الديهايد ومعالم النطم فقمد ظشماره النتمائ إلما حصمول نقمث معنموي 0.05>P في النسبة المئوية للنط المتحركة ودرجة نشاط النط والنسبة المئويمة لعيوشمية النطم وااداد هملا المنقث بايماد فتمر الح ممن سمماعتين وظربمم سمماعاه لعينمماه منممي مر مما وهممن النطمم وابي مماض المنممي فممي حمين لمموح ايمماد يممر معنوية 0.05<P في تركيا المالونداي الديهايد باياد فتراه الح ن 24