IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (1) 2010 Effects of Licorice Extract on Sperm Motility of Chilled Stored Ram Semen A.K.Mahdi Department of Biology, College of Education Ibn Al-Haitham, University of Baghdad Abstract This study was designed to investigate the effects of licorice extract (Glycyrrhiza glabra L.) addition to semen diluters on ram sperm progressive motility during storage at 5 ˚C for 72 h. Semen was collected from 3 proven Awassi rams. Licorice extract powder was added at levels of 1, 5, 10, 50 and 100 µg per ml. of diluter. Diluter containing no licorice extracts served as control (0).Progressive motility was estimated subjectively after dilution (0h), and at 24, 48, and 72 h of storage. The experiment was replicated 2 times with egg yolk-tris (EYT) diluter and 2 times with yolk- glucose-citrate diluter. Progressive motility increased significantly (p < 0.01) in levels of licorice extract 1, 5, 10, 50 and 100 µg / ml in both diluters, during all storage periods. The means of progressive motility were 72.5 ± 1.02 %, 72.08 ± 1.05, 70.90 ± 2.05 % and 66.25 ± 3.15 % respectively, compared to the control (0) 61.45 ± 16.2 %. Levels 1, 5 and 10 µg /ml were superior (p < 0.01) to levels 50 and 100 µg /ml. In conclusion, the addition of licorice extract to the diluter improved ram sperm progressive motility during cooled storage at 5 ˚C. Key words: Licorice extract; diluter; ram semen; progressive motility. Introduction Licorice is the name applied to the roots and rhizomes of Glycyrrhiza species and has been used for medicinal purposes for at least 4000 years [1]. Glycyrrhiza glabra L. is one of the very important nutraceuticals, contains some 400 bioactive phytochemicals [2], and has many documented bioactivities such as: steroid like activity [3], powerful antioxidants activity [4], antibacterial activity [5] and antiviral activity [6]. In traditional herbal medicine, licorice was considered as a natural source of sex hormones and used to: strengthen female reproductive system [7], treat some women's sterility cases in Japan and China [8,9], strengthen male reproductive system, improve sperm count as well as semen viscosity in Ayurvedic medicine [10], and improve erection [11,12]. Our previous study results indicated that treatment with licorice improved reproductive performance of ram lambs and rams [13]. Many additives were used to improve preserved semen quality such as: dyes [14], caffeine [15], sugars [16], aromatic compounds [17], chelating agents [18], antioxidants [19], selenium [20], seminal plasma [21] and soybean [22]. Licorice contains many phytochemicals which may have ameliorating effects on semen quality , so this study was designed to investigate the possible effects of licorice extract addition to the diluter on sperm motility of chilled stored ram semen. Material and Methods Two types of diluters were used, egg yolk- tris (EYT) [23] and yolk- glucose-citrate [24]. Licorice extract powder (Glycyrrhiza glabra L.) purchased from local company (Al-Ahliah Company for Aromatic Odors & Flavorings Production Ltd., Baghdad, Iraq) was added at IBN AL- HAITHAM J. FOR PURE & APPL. SCI . VOL.23 (1) 2010 levels of 1, 5, 10, 50 and 100 µg per ml. of diluter. Diluter containing no licorice extracts served as control (0). Diluters were prepared the day prior to use, allowing large participle to settle overnight at 5˚C, so that the supernatant could be used. Before use, each diluter was warmed to 37 ˚C. Fresh semen was collected by an artificial vagina from Awasi rams. Semen of 3 proven rams was pooled, mixed and kept in a water bath at 37 ˚C. Semen was diluted 1:10 (vol. /vol.) to different diluters, then gradually cooled by putting the tube of diluted semen in about 500 ml of 37 ˚C water containers and placed in a 5 ˚C refrigerator for 2 hours to reduce cold shock [24], then stored at 5 ˚C for 72 h. Progressive motility was estimated subjectively after dilution(0h), and at 24, 48, 72 h of storage, by diluting a drop of semen with 0.9 % sodium chloride solution in a warm slide, mounting it with cover slip and examining it under a microscope at X 400 magnification, using a 100-point scale for linear movement [25]. The data were expressed in percentage of total cells. M otility estimations were performed from 5 different fields in each sample by the same person throughout the study; the mean value averaged from 5 successive estimations was used as the final motility score. The experiment was replicated 2 times with egg yolk-tris (EYT) diluter and 2 times with yolk- glucose-citrate diluter. The Statistical Analysis System [26] general linear model was used to analyze the data. Differences among treatment means were compared for statistical significance, us ing Duncan's multiple range test [27]. Results Progressive motility increased significantly (p < 0.01) in levels of licorice extract 1, 5, 10, 50 and 100 µg / ml in both diluters, during all storage periods. The means of progressive motility were 72.5 ± 1.02 %, 72.08 ± 1.05, 70.90 ± 2.05 % and 66.25 ± 3.15 % respectively, compared to the control (0) 61.45 ± 16.2 %. (fig1). Levels 1, 5 and 10 µg /ml were superior (p < 0.01) to levels 50 and 100 µg /ml (fig1). Diluter type had a significant effect (p < 0.01) on sperm motility. Overall the percentage of motile sperm in EYT diluter (66.48 ± 1.21 %) was higher than that in yolk-glucose citrate diluter (64.37± 1.44 %). Sperm motility tended to decline significantly (p < 0.01) as the length of storage period increased. The means of progressive motility were 80.00 ± 2.04 % after dilution (0h), 68.75 ±3.15 % 61.25 ± 4.27% and 50.62 ±4.61 %, at 24, 48 and 72 h after cooling, respectively. Discussion Although the history of licorice is as old as the history of medicine and of confection [28] and references to licorice date back to approximately 2500 B.C on Assyrian clay tablets and Egyptian papyri [29], this study, to the best of our knowledge, is the first indication for a positive effect of licorice (Glycyrrhiza glabra L.) on sperm motility of chilled stored ram semen. Sperm motility is regarded as a manifestation of sperm functional competence [30] and licorice extract improved (p < 0.01) progressive motility especially in low levels (1, 5, 10 µg /ml diluter). Progressive motility is the most important individual quality test, because fertility is highly correlated with number of motile sperm inseminated [24]. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (1) 2010 The precise role of the components of diluters (sugars, proteins, a range of additives, etc.) in preserving the integrity and the fertilizing potential of spermatozoa is still far from being understood, and the composition of the diluents has been improved by more or less empirical studies [31,32,33]. Licorice may be considered as a versatile additive. First, an important reason for the decrease in motility during the storage of semen is the formation of lipid peroxides from oxygen radicals [34]. The sperm plasma membrane contains a high amount of unsaturated fatty acids and is therefore particularly susceptible to peroxidative damage [35]. The lipid peroxidation destroys the structure of the lipid matrix in the membranes of spermatozoa, and it is associated with a loss of motility and membrane integrity [36]. Semen contains appreciable amounts of antioxidants that balance lipid peroxidation and prevent excessive peroxide formation [37] but the endogenous antioxidative capacity of semen may be insufficient during storage or dilution [38]. The addition of antioxidants is well known to improve the viability and motility of liquid storage or cryopreserved ram sperm cells [19, 39]. Licorice has powerful antioxidant activity, it is contains seven antioxidant compounds, four isoflavans, two chalcones and an isoflavone [4], and these compounds were shown to be effective to protect biological systems against various oxidative stresses [40] via a mechanism involving scavenging of free radicals [41]. Second, licorice contains many sugars such as fructose and sucrose [11]. Fructose is one of the principle energy substrate for spermatozoa [24] and an activator factor of mammal spermatozoa [42], sucrose are reported to be effective in stabilizing sperm membrane bilayers during storage [43]. Third, Licorice has antimicrobial activity, and the antimicrobial agents are routinely added to semen diluters to control many semen organisms which are pathogenic and compete with sperm for nutrients and produce metabolic by-products that have an adverse effect on livability of the sperm [24]. The decrease in sperm motility at high levels of licorice extract in this study (50 and 100 µg /ml) is possibly caused by high osmolality ; hyper osmotic environment can inhibit sperm motility [44]. Further researches are needed to design an isotonic diluter with high level of licorice extract. Overall progressive motility was better in EYT. Different diluents for semen have been used to improve fertility rate. EYT extender was recommended by many researchers and commonly used for artificial insemination [23, 45]. Over storage period, sperm motility declined significantly (p < 0.01) probably due to the accumulation of the toxic products of sperms metabolism [38]. The study findings may contribute to the recent attempts to design defined semen diluter and move away from animal-based cryoprotectants, which may pose hygienic risks and are difficult to standardize [46]. Finally there are several factors affecting the phytochemistry of the licorice root such as geographical location, soil condition, time of harvesting and the environmental factors [47] and this should be considered when applying such treatment widely. In conclusion, the addition of licorice extract to the diluter improved ram sperm progressive motility during cooled storage at 5 ˚C. References 1. Nomura, T.; Fukai, T. and Akiyama, T .(2002) Chemistry of phenolic compounds of licorice (Glycyrrhiza species) and their estrogenic and cytotoxic activities. Pure Appl. Chem., 74:1199–1206 2. Duke, J. (1995) Phytochemical constituents in licorice. In: Nutraceticals: Designer Foods III: Garlic, Soy and Licorice .Ed. P.A. Lachance .Food and Nutrition Press, Inc., USA Chap.26, pp. 259-264. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (1) 2010 3. Kumagi, A.; Yano, M.; Otomo, M. and Takeuchi, K. (1957) Study on the corticoid – like action of glycyrrhizin and mechanism of its action .Endocrinol.Jpn.4:17-27. (Abstr.) 4. Vaya, J.; Belinky, P.A and Aviram, M. (1997) Antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidatives capacity toward LDL oxidation. Free Radic. Bio. Med., 23:302-313. 5. Hatano, T.; Shintani, Y.; Aga, Y.; Shiota, S.; Tsuchiya, T. and Yoshida, T. (2000) Phenolic constituents of licorice.VII: structures of glicophenone, and glicoisoflavone and effects of licorice phenolic on methicillin-resistant Staphylococcus aureus. Chem. Pharm. Bull., 48: 1286-1292. (Abstr.). 6. Jeong, H.G. and Kim, J.Y. (2002) Induction of inducible nitric oxide synthase expression by 18b-glycyrrhetininc acid in macrophage. FEBS Lett. , 513:208-212. (Abstr.). 7. Paulien, G.B. (1995).The Divine Philosophy and Science of Health and Healing. Teach Services, Inc., USA., p .366. 8. Yaginuma, T.; Ismumi, R.; Arai, T. and Kawabata, M. (1982) Effect of traditional herbal medicine on serum testosterone levels and its induction of regular ovulation in hyperandrogenic and oligomenorrhic women. Act. Obstet. Gynaec.Jpn., 34:939-944. 9. Ostrzenski, A. (2001) Gynecology: Integrating Conventional, Complementary, and Natural Alternative Therapy. Lippincott Williams & Wilkins, USA., p.147. 10. Moharana, D. (2008). Shatavari, Jastimadhu and Aswagandha the Ayurvedic therapy. Orr. Rev., 7-8:72-77. 11. Ross, I.A. (1999) Glycyrrhiza glabra. In: Medicinal Plants of the World: Chemical Constituents, Traditional, and Modern Medicinal Uses. Humana Press, USA.Chap.11, pp.191-227. 12. Duax, W.L. (2000) Personal communication. Hauptman –Woodward Medical Research Institute, Buffalo, N.Y, USA. 13. Mahdi, A.K. (2000) Effect of licorice extract on reproductive performance of Awassi rams. M .Sc. thesis, College of Agriculture, Baghdad University. 14. Phillips, P.H. (1945) A practical method of coloring semen for identification purpose .J. Dairy Sci., 28: 843-844. (Abstr.) 15. Garbers, D.L. First, N.L., Sullivan, J.J., and Lardy, H.A. (1971) Stimulation and maintenance of ejaculated bovine spermatozoon respiration and motility by caffeine. Bio. Reprod., 6:336-339. 16. Abdelhakeam, A. A., Graham, E. F., and Chaloner, E.M. (1991) Studies on the absence of glycerol in unfrozen and frozen ram semen. Development of an extender for freezing: effects of osmotic pressure, egg yolk levels, types of sugar and method of dilution .Cryobiology, 28: 43-47 17. Bamba, K. and Miyagawa, N.(1992) Protective action of aromatic compounds against cold-shock injuries in boar spermatozoa. Cryobiology, 29: 533-536. (Abstr.) 18. Nishimura, K. (1993) Effects of calcium ions on the malate –aspartate shuttle in slow cooled boar spermatozoa. Biol.Reprod., 49:537-543. (Abstr.) 19. Maxwell, W.M. and Stojanov, T. (1996) Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod.Fertil.Dev., 8: 1013-1020. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (1) 2010 20. Seremak, B. ,Udala, J., and Lasota, B .(1999) Influence of selenium on ram semen freezing quality . Electr.J.of Pol. Agri. Univ., 2: 110-117. 21. Ghaoui, R.E.H; Gillan, L.; Thomson, P.C.; Evans, G. and Maxwell, W.M.(1996) Effect of seminal p lasma fractions from entire and vasectomised rams on the motility characteristics, membrane status and in vitro fertility of ram spermatozoa. J. Androl., 28:109-122. 22. Fukui, Y.; Kohno, H.; Togari, T.; Hiwasa, M. and Okabe, K.(2008) Fertility after artificial insemination using a soybean-based semen extender in sheep. J. Reprod. Dev., 54:286-289. 23. Liu, Z. and Foote, R. (1998) Osmotic effects on volume and motility of bull sperm exposed to membrane permeable and non-permeable agents. Cryobiology, 37: 207- 218. 24. Bearden, H.J, and Fuquay, J.W.(1997) Applied Animal Reproduction .4th edn. Prentice Hall, USA. , pp.133-141, 158-191. 25. Walton, A. (1933). The Technique of Artificial Insemination. Imperial Bureau Anim. Genetics. Oliver and Boyd, Edinburgh.UK. 26. SAS. (2001) SAS User's Guide: Statistics. SAS Inst. Inc., N.C., USA. 27. Duncan, D.B.(1955).Multiple range and multiple F tests. Biometrics, 11:1-42. 28. Peter, S. V., and Testa, L. C.A. (1995) Phytochemistry of licorice horticultural and processing procedures. In: Nutraceticals: Designer Foods III: Garlic, Soy and Licorice .Ed. P.A. Lachance .Food and Nutrition Press, Inc., USA. Chap.25, 243-257. 29. Braun, L., and Cohen, M. (2007) Licorice .In: Herbs & Natural Supplements .2 nd edn. Elsevier Australia. pp. 786-807. 30. Scott, M.A. (2000).A glimpse at sperm functions in vivo: sperm transport and epithelial interaction in the female reproductive tract. Anim. Rep. Sci., 60-61: 337- 348. 31. Polge, C.;Smith, A.U. and Parks, A.S. (1949) Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature, 164:666–669. (Abstr.) 32. Gebauer, M. R.; Pickett, B. W.; Komarek, R. J. and Gaunya, W. S. (1970).Motility of bovine spermatozoa extended in defined diluents. J. Dairy Sci., 53: 817-823. 33. Bergeron, A. and Manjunath, P.(2006) New insights towards understanding the mechanisms of sperm protection by egg yolk and milk. Mole.Rep.Dev., 73: 1338- 1344 (Abstr.) 34. Aurich, J. E.; Shonder, U.; Hoppe, H. and Aurich, C. (1997) Effect of antioxidants on motility and membrane integrity of chilled-stored stallion semen. Theriogenology, 48: 185-192. 35. Griveau, J.F.; Dumont, E.; Renard, P.; Callegari, J.P. and Lelannou, D.(1995)Reactive oxygen species, lipid peroxidation and enzymatic defense systems in human spermatozoa. J. Reprod. Fertil., 103: 17-26. 36. Sharma, R.K. and Agarwal, A. (1996) Role of reactive oxygen species in male infertility . Urology, 48: 835-850. 37. Lewis, S.E.M.; Sterling, E.S.L. and Young, L.S.(1997) Comparison of individual antioxidants of semen and seminal plasma in fertile and infertile men. Fertil.Steril., 67: 142-147. 38. Maxwell, W.M. and Salamon, S. (1993) Liquid storage of ram semen. Reprod.Fertil. Dev., 5: 613-638. 39. Sanchez-Partida, L.G.; Setchell, B.P. and Maxwell, W.M.(1997) Epididymal compounds and antioxidants in diluents for the frozen storage of ram spermatozoa. Reprod. Fertil. Dev., 9: 689-696. IBN AL- HAITHAM J. FOR PURE & APPL. SCI. VOL.23 (1) 2010 40. Haraguchi, H.; Ishikawa, H.; Mizutani, K.; Tamura, Y. and Kinoshita, T. (1998) Antioxidative and superoxide scavenging activities of retrochalcones in Glycyrrhiza inflate. Bioorg. Med. Chem., 6: 339-347. 41. Fuhrman, B.; Buch, S.; Vaya, J.;Belinky, P.A.; Coleman, R.; Hayek, T. and Aviram, M. (1997) Licorice extract and its major polyphenol glabridin protect low-density lipoprotein against lipid peroxidation: in vivo and ex vivo studies in human and in atherosclerotic apolipoprotein E-deficient mice. Am. J. Clin. Nutr., 66: 267-275. 42. Rigau, T.; Camprubi, M.; Badia, J.; Ballester, J. and Palomo, M.J. (2000) Different effects of glucose and fructose on energy metabolism in dog sperm from fresh ejaculates. 14 th International Congress on Animal Reproduction, 2:24. 43. Chen, Y.; Foote, R.H. and Brockette, C. (1993) Effect of sucrose, trehalose, taurine and blood serum on survival of frozen bull sperm .Cryobiology, 30:423-431. 44. Pommer, A.C. Rutllant, J. and Meyers, S. A. (2002). The role of osmotic resistance on equine spermatozoa function. Theriogenology, 58: 1373-1384. 45. Evans, G. and Maxwell, W.M. (1987) Salamon’s Artificial Insemination of Sheep and Goats. Sidney, Butterworth, Australia, pp. 66-76. (Cited by Maxwell & Salamon, 1993). 46. Ball, P.J.H., and Peteres, A.R. (2004) Reproduction in Cattle.3 rd edn. Blackwell Publishing, UK ., p. 131. 47. Lutomski, J., Nieman, C., and Fenwick, G.R. (1991) Licorice, Glycyrrhiza glabra L. biological properties. Herba Polonica, 37:163-178. 2010) 1( 23مجلة ابن الھیثم للعلوم الصرفة والتطبیقیة المجلد لسائل افي لحیامن لالحركة الفردیة قیم في مستخلص عرق السوس تأثیر المنوي المبرد للكباش أحمد قاسم مھدي ابن الھیثم ، جامعة بغداد- قسم علوم الحیاة ، كلیة التربیة خالصةال ت إلى (.Glycyrrhiza glabra L)مستخلص عرق السوس إضافة تأثیرمت هذه التجربة لدراسة صم مخففا جمع .ساعة 72ة مد ˚م 5لحیامن الكباش خالل حفظ السائل المنوي بدرجة الحركة الفردیة على قیم السائل المنوي و 50 و 10و 5و 1مستویات رق السوس بمستخلص ع أضیف.السائل المنوي من ثالثة من كباش العواسي المختبرة . )0(مجموعة مقارنة محتوي على مستخلص عرق السوس الغیر استعمل المخخف .مل من المخفف / مایكروغرام 100 كررت . ˚م 5ساعة من الحفظ بدرجة 72و ، 48،و 24،و بعد ، (0h) ركة الفردیة بعد التخفیف تم تقدیر قیم الح صفار -الكلوكوز–السترات ومرتین باستعمال مخفف ) (EYTصفار البیض -ال مخفف الترس التجربة مرتین باستعم مخففة والمضاف (p < 0.01)قیم الحركة الفردیة معنویا ازدادت. البیض مستخلص إلیهافي عینات السائل المنوي ال كانت . الحفظ مددخالل كل ن كال المخففی في مل/ مایكروغرام 100و 50 و 10و 5و 1عرق السوس بمستویات % 3.15± 66.25و % 2.05± 70.90 و% 1.05± 72.08و % 1.02± 72.5م الحركة الفردیة قی متوسطات في مل / مایكوغرام 10و 5و 1وتفوقت المستویات . %16.2± 61.45) 0(على التوالي مقارنة بمجموعة المقارنة نستنج من نتائج هذه . مل في قیم الحركة الفردیة / مایكوغرام 100و 50ن یستویعلى الم (p < 0.01) المخفف معنویا م الحركة الفردیة إلىمستخلص عرق السوس إضافةالدراسة ان ة الحفظ مدلكباش خالل لحیامن االمخفف قد حسن من قی . ˚م 5المبرد بدرجة