Changes in Sperm Parameters of Adult Male Rabbits by Phenol Extract of Lepidium Sativum Seeds

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1 Chnges in Sperm Prmeters of Adult Mle Rits y Phenol Extrct of Lepidium Stivum Seeds Nd Sd Nji Deprtment of Generl Sciences, College of Bsic Eduction, University of Bylon, PO ox 4, Irq E-mil of the corresponding uthor: Dr_n_sd_Alte@yhoo.com Fris Nji Aood Deprtment of Biology, College of Science, University of Bylon, PO ox 4, Irq Astrct This reserch included the impct of phenol extrct of Lepidium stivum seeds in sperm prmeters of dult mle rit. The In Vivo study included the estimtion of the Medium Effect Dose (MED 50 ) for phenol extrction y using Dose-Response Curve. The men of MED 50 ws 36.1 mg /kg ody weight (.w.) of phenol. The results showed significnt increse (P in testiculr sperm concentrtion, epididymus sperm concentrtion nd in the sperm count per gm of the testis, sperm motility percent, grde ctivity, sperm viility percent,nd norml sperm morphology percent of epididymis cudl t MED 50 of phenol. The results revel tht supplement with low doses of phenol could e enhnce rit fertility. Keywords:Lepidium stivum, seeds, phenol, soxhlet device, rits, sperm prmeters 1. Introduction The Lepidium stivum (fmily Brssiccee) is ntive shru (Gill & McLeod 1980). It is usully cultivted for its leves, which re used in sld, sndwiches etc. (Lefroy Vlley crries (LVC) 2013). The leves nd seedpods hve peppery tste, nd hve een reported to hve enormous iologicl ctivities (Srikmi & Ynmz 2011). It is documented to possess, phenol compounds, tocopherol, nitrogen compounds, terpenoids, nd some other endogenous metolites, which re rich in ntioxidnt ctivity (Mund et l. 2011). nd Ahmed et l. (2013) reveled tht Lepidium stivum seeds with high nutritionl vlue cn e exploited s functionl food ingredient. Antioxidnts re vitl sustnces which possess the ility to protect the ody from dmges cused y free rdicl-induced oxidtive stress nd vriety of free rdicl scvenging ntioxidnts re found in dietry sources like fruits, vegetles nd te (Souri et l. 2008). Phenolic compounds re lrge group of phytochemicls widespred in the plnt kingdom. Depending on their structure they cn e clssified into simple phenols, phenolic cids, hydroxyl cinnmic cid derivtives nd flvonoids (Crte et l. 2012). To dte, there re pproximtely 100 pulictions on this topic, which highlight the eneficil effects of this ntioxidnt on viility, memrne integrity nd motility of spermtozo of different species. 2. Mterils nd Methods 2.1 Preprtion of Extrcts Grden cress seeds (Lepidium stivum L.) were otined from the locl mrket in Hill City, Irq. The seeds were clened nd rendered free of dust, then stored in polyethylene gs in the refrigertor until used. For phenol extrction, grden cress seeds were crushed, y using household mill (Brun, Germny), nd then 1 g smple ws extrcted with 25mL of (80% methnol : 10% Distilled wter) in wter th t 70 C for 50 minutes. The extrct ws filtered out nd evported to dryness y oven t 45 0 C for 24 hour (Hrorn 1984). 2.2 Experimentl nimls Twenty (20) New Zelnd White mle rits ged 4 months nd vergely weighing grm nd put in cge under control of wter, diet, light durtion (12hour light-12hour drk). These nimls were divided into 4 groups (5 nimls for ech group), control group ws treted orlly with distilled wter nd experimentl groups were treted orlly with 32, 64, nd 96 mg/kg.w. of phenol extrct nd for 50 dys dily with these concentrtion. The nimls were seduced fter period end of experience with chloroform then the epididymis cudl ws excised to clculte the rte of sperm concentrtion in epididymis cudl, sperm motility percent, nd grde ctivity, nd find MED 50 for ech prmeter, nd then clculting the verge of the ove doses ffecting. Effective dose of extrct ws used fter its ppointment in the first experiment to know its impct on reproductive efficiency of rits, using 10 mle rit, nd these nimls were divided into 2 groups, nd y 5 nimls per group nd treted s descried elow: Group A: control group, treted orlly with distilled wter for 50 dys. Group B: phenol group, treted orlly with 33.6 mg/kg.w. of extrct for 50 dys. The nimls hve een weighed fter the expirtion of the effective dosge using the lnce, nd they were seduced fter period end of experience with chloroform nd ll of the left testicle nd left epididymis were used to study of sperm prmeters. 17

2 2.3 Sttisticl nlysis Results were presented s Men ± S.E. Sttisticl nlysis ws done using SPSS nd ws conducted using one wy ANOVA to compre the mens, F-test, T-test, correltion coefficient, nd use the Lest Significnt Difference (LSD) in the comprison etween the results nd lso regression nlysis nd regression coefficient. Different letters ws considered significnt (P<0.05) (Rndolph & Ciminer 1985; AL-Rwi 2000). 3. Results In Vivo Experiments, the tretment of nimls with 32 nd 64 nd 96 mg/kg.w. of phenol extrct led to get significnt increse (P<0.05) in ech of sperm concentrtion of epididymis cudl nd sperm motility percent for the treted groups, compred with the control group. It hs een oserved significnt increse (P<0.05) in sperm motility percent with progressive movement of the epididymis cudl for the groups treted with 64 nd 96 mg/kg.w., compred with the control group nd the group treted with 32mg/kg.w. (Figure 1, 2 nd 3). Figures 4,5 nd 6 show positive liner reltionship etween esclting doses of phenol extrct nd sperm concentrtion, sperm motility percent, nd sperm motility percent with progressive movement of the epididymis cudl where the correltion coefficient were r = 0.865, 0.789, nd 0.874; nd the regression eqution liner stndrd s follows: y = (x), y = (x), y = (x) of the ove prmeters, respectively. The results reveled significnt increse in the three prmeters of the epididymis cudl, so the vlues of MED50 were , , nd mg/kg.w. of phenol extrct of the three criteri respectively. The verge ws clculted for these vlues in order to e used in susequent experiment. Accordingly, the vlue is equl to 36.1mg/kg.w.of phenol extrct s shown in the tle (1). 4. Discussion The results reveled tht phenol extrct of Lepidium stivum seeds led to significnt increse in the rte of sperm concentrtion in epididymis cudl, due to its ntioxidnt nd kept the ody from vrious chemicl rections of free rdicls, which poison the cells (Drgsted 2003). The resercher (Mhneem et l. 2011) hs pointed out tht the increse in the sperm concentrtion of rts treted orlly t 1.2 g/kg.w. of honey for 28 dys, returning to the presence of some ntioxidnts, including phenols, flvonoids, vitmin A, E nd ctlse enzyme. Our results showed tht the dosge of dult mle rits with influentil concentrtion of phenol extrct; led to significnt increse in sperm motility percent, grde ctivity, nd sperm viility percent of epididymis cudl. This might return to the effect of Lepidium meyenii roots, which relted to the Rshd plnt, nd to the ssocition the romtic compounds, including Isothiocyntes with Aphrodisic ctivity, or to link phenol compounds with ntioxidnts ctivity, or possily due to the influence of the Blck Mc long the eighth stge of seminiferous epithelil tuules (Gonzles et l. 2006; Yucr et l. 2008; Singh et l. 2011). The increse in norml sperm morphology percent of rits epididymus cudl treted with effect dose of phenol extrct in the current study, my e return to the use of the high concentrtion of phenol, which my interfere in the process of spermtids formtion, which led to dmge in the process of the sperm hed configuring, forming norml sperm, nd perhps otined s result of genetic overlp nd cuse normlities in sperm. This result is consistent with the results of Michlowicz nd Dud (2007), due to oth of phenolic compounds nd Ctechol hve hrmful toxics, nd toxicity of phenol my return to the toxic non-specific, relting to Hydrophoocity, the soluility of phenol in the prts of the cell, nd thus the possiility of the interction of these compounds with cell nd structures of tissues. Both of Ctechol nd Hydroquinone Inhiits the effectiveness of the rionucleotide reductse enzyme, which is involved in the mnufcture of DNA (McCue et l. 2003). And toxicity of phenols my e ck to the roots of Phenoxy nd to toxicity resulting from metolites (Zitk et l. 2011). The interct of phenolic cids with proteins led to chnge in composition of cronyl groups nd oxidtion of thiol groups (Lieniec & Gryelk 2006). The phenolic compounds own the property of production of hydrogen peroxide in the presence of some metls (Crte et l. 2011), where hydrogen peroxide is kind of rective oxygen species, due to its Interction etween the root of the hydroxyl nd chins unsturted ftty cid in the cell memrne; leding wht is known s the lipid peroxidtion (Vermerris & Nicholson 2006). 5. Conclusion Under the light of this reserch, it is concluded tht phenol extrct of Lepidium stivum seeds is the most importnt ntioxidnt. Our results reveled significnt increse in testiculr sperm concentrtion, epididymus sperm concentrtion nd in the sperm count per gm of the testis, sperm motility percent, grde ctivity, sperm viility percent, nd norml sperm morphology percent of epididymus cudl. According to the results it cn e concluded tht phenol extrct improves some prmeters of sperm. 18

3 References Ahmed, M.G., Azz, A.M. & He, E.E. (2013), Chemicl, Nutritionl nd Biochemicl Studies of Grden Cress Protein Isolte, NtSci 11 (2):8-13. AL-Rwi, k.m. (2000), Introduction to Sttistics, 2 nd.ed, College of Agriculture nd Forestry. Mosul University. Crte, M.E., Frncisco, M., Soengs, P. & Velsco, P. (2011), Phenolic Compounds in Brssic Vegetles, Molecules 16: Crte, M.E., Frncisco, M., Soengs, P. & Velsco, P. (2011), Phenolic Compounds in Brssic Vegetles, Molecules 16: Drgsted, L.O.(2003), Antioxidnt Actions of Polyphenols in Humns, Int. J. Vitm. Nutr. Res. 73(2): Gill, V. & McLeod, A.J. (1980), Studies on Glucosinolte Degrdtion in Lepidium Stivum L. Seed Extrcts, Phytochem 19: Gonzles, C., Ruio, J., Gsco, M., Nieto, J., Yucr, S. & Gustvo, F.G. (2006), Effect of Short-Term nd Long-Term Tretments with three Ecotypes of Lepidium meyenii (MACA) on Spermtogenesis in Rts, Journl of Ethnophrmcology 103: Hrorn, J.B. (1984), Phytochemicl methods: Aguide to Modern Techniques of Plnts Anlysis, 2 nd.ed London, New York, Chpmn nd Hll. Lieniec, M. & Gryelk, T. (2006), Study of Interctions etween Phenolic Compounds nd H 2 O 2 or Cu(II) ions in B 14 Chinese Hmster Cells, Cell Biology Interntionl 30: Lefroy Vlley crries (LVC) (2013), Her Rnge. Austrli/New Zelnd, 1300 lefroy WWW. lefroyvlley.comout. Mhneem, M., Sulimn, S.A., Jfr, H., Sirjudeen, K.N.S., Ismil, Z.I.M. & Islm, M.N. (2011), Effect of Honey on Testiculr Functions in Rts Exposed to Cigrette Smoke, ApiProduct & ApiMedicl Science 3 (1): McCue, J., Lzis, S., Cohe, N.J., Modino, J. & Freed, B. (2003), Hydroquinone nd Ctechol Interfere with T Cell Entry nd Progression through the G(1) Phse, Mol. Immunol.39:995. Michłowicz, J. & Dud, W. (2007), Phenols - Sources nd Toxicity, Polish J. of Environ. Stud. 16(3): Mund, F.N., Bouyed, J., Djilni, A., Yo, C., Soulimni, R. & Dicko, A. (2011), Chemicl Composition nd, Cellulr Evlution of the Antioxidnt Activity of Desmodium dscendens Leves, Evidence-Bsed Complementry & Alterntive Medicine 2011:1-9. Rndolph, L.K. & Ciminer, J.L. (1985), Sttistics In : Remington ' s Phrmceuticl Sciences, 17 th.ed., Gennro, A.R. (ed) Mck Srikmi, G. & Ynmz, R. (2011), Effects of Cultivr nd Developmentl Stge on Glucosinoltes in Grden Cress (Lepidium Stivum L.), Medicinl Plnts Reserch 5(17): Singh, K.J., Singh, R., Singh, S., Singh, K., Frswn, M. & Singh, V. (2011), Aphrodisic Agents from Medicinl Plnts: A review, Chem. Phrm. Res. 3(2): Souri, E., Amin, G., Frsm, H. & Brzndeh, T.M. (2008), Screening of Antioxidnt Activity nd Phenolic Content of 24 Medicinl Plnt Extrcts, DARU 16 (2): Vermerris, W. & Nicholson, R. (2006), Phenolic Compound Biochemistry, Pulished y Springer. P.O. Box 17, 3300 AA Dordrecht, The Netherlnds. PP: Yucr, S., Gsco, M., Ruio, J., Nieto, J. & Gonzles, G.F. (2008), Effect of Different Frctions from Hydrolcoholic Extrct of Blck Mc (Lepidium meyenii) on Testiculr Function in Adult Mle Rts, Fertility nd Sterility 89(5): Zitk, O., Sochor, J., Rop, O., Sklickov, S., Sorov, P., Zehnlek, J., Beklov, V., Krsk, B., Adm, V. & Kizek, R. (2011), Comprison of Vrious Esy-to-Use Procedures for Extrction of Phenols from Apricot Fruits, Molecules 16:

4 C C Figure 1. Impct of Esclting Doses of Phenol Extrct in the Rte of Sperm Concentrtion in the Epididymis Cudl (Million/Ml) of Mle Rits c Figure 2. Impct of Esclting Doses of Phenol Extrct in the Sperm Motility Percent in the Epididymis Cudl (Million/Ml) of Mle Rits Figure 3. Impct of Esclting Doses of Phenol Extrct in the Sperm Motility Percent with Progressive Movement in the Epididymis Cudl (Million/Ml) of Mle Rits 20

5 Figure 4. Vrition of sperm concentrtion rte with esclting doses of phenol extrct in mle rits Figure 5. Vrition of Sperm Motility Percent with Esclting Doses of Phenol Extrct in Mle Rits 21

6 Figure 6. Vrition of Sperm Motility Percent of Progressive Movement with Esclting Doses of Phenol Extrct in Mle Rits Tle 1. Medin Effective Dose 36.1 Mg/Kg.w. of Phenol Extrct in Sperm Prmeters of the Testis nd Epididymis Cudl of White Mle Rits After Orl Dosing for 50 Dys Groups Control Effect Dose 36.1 Mg/Kg/Dy of Phenol Extrction Sperm Prmeters Testiculr Sperm Concentrtion (Million/Ml) ± ± Epididymis Cudl Sperm Concentrtion (Million/Ml) ± ± Sperm Count Rte Per Gm of the Testis ± ± Sperm Motility Percent of Epididymis Cudl ± ± Sperm Motility Percent with Progressive Movement ± ± (+) of the Epididymis Cudl Sperm Motility Percent with Progressive Movement (c) ± ± of the Epididymis Cudl Sperm Motility Percent with Progressive Movement (d) of the Epididymis Cudl ± ± Anorml Sperm Morphology Percent of Epididymis ± ± Cudl Sperm Viility Percent of Epididymis Cudl ± ±