IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (1) 2010 Effects of Licorice Extract on Sperm Motility of Chilled Stored Ram Semen A.K.Mah di Departme nt of Biology, College of Education Ibn Al-Haitham, Unive rsity of Baghdad Abstract This st udy was designed to inv estigate the effects of licorice extract (Glycyrrhiza glabra L.) addition to semen diluters on ram sp erm p rogressive motility during st orage at 5 ˚C for 72 h. Semen was collected from 3 p roven Awassi rams. Licorice extract p owder was added at levels of 1, 5, 10, 50 and 100 µg p er 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 st orage. The exp eriment was replicated 2 times with egg y olk-tris (EYT) diluter and 2 times with y olk- glucose-citrate diluter. Progr essive motility increased significantly (p < 0.01) in levels of licorice extract 1, 5, 10, 50 and 100 µ g / ml in both diluters, during all st orage p eriods. The means of p rogr essive motility were 72.5 ± 1.02 %, 72.08 ± 1.05, 70.90 ± 2.05 % and 66.25 ± 3.15 % resp ectively, comp ared to the control (0) 61.45 ± 16.2 %. Levels 1, 5 and 10 µg /ml were sup erior (p < 0.01) to levels 50 and 100 µg /ml. In conclusion, the addition of licorice extract to the diluter imp roved ram sp erm p rogressive motility during cooled st orage at 5 ˚C. Key words: Licorice extract; diluter; ram sem en; progressive mo tility. Introduction Licorice is t he name app lied to t he roots and rhizomes of Glycyrrhiza sp ecies and has been used for medicinal p urp oses for at least 4000 y ears [1]. Glycyrrhiza glabra L. is one of the very imp ortant nutraceuticals, contains some 400 bioactive p hytochemicals [2], and has many documented bioactivities such as: st eroid like activity [3], p owerful antioxidants activity [4], antibacterial activity [5] and antiviral activity [6]. In traditional herbal med icine, licorice was considered as a natural source of se x hor mones and used to: strengthen female reproductive sy stem [7], treat some women's sterility cases in Jap an and China [8,9], strengthen male reproductive sy stem, imp rove sp erm count as well as semen viscosity in Ay urvedic medicine [10], and improve erection [11,12]. Our p revious st udy results indicated that treatment with licorice imp roved reproductive p erformance of ram lambs and rams [13]. M any additives were used to imp rove p reserved semen quality such as: dy es [14], caffeine [15], sugars [16], aromatic compounds [17], chelating agents [18], antioxidants [19], seleniu m [20], seminal p lasma [21] and soy bean [22]. Licorice contains many p hy tochemicals which may have amelioratin g effects on semen quality , so this study was designed to invest igate the p ossible effects of licorice extract addition to t he diluter on sp erm motility of chilled stored ram semen. Material and Methods Two typ es of diluters were used, egg y olk- tris (E YT) [23] and y olk- glucose-citrate [24]. Licorice extract p owder (Glycyrrhiza glabra L.) p urchased from local company (Al-Ahliah Company for Aromatic Odors & Flavorings Production Ltd., Baghdad, Iraq) was added at IBN AL- HAITHAM J. FOR PURE & APPL. S CI . VOL.23 (1) 2010 levels of 1, 5, 10, 50 and 100 µg p er ml. of diluter. Diluter containing no licorice extracts served as control (0). Diluters were p repared the day p rior to use, allowing lar ge p articip le to settle overnight at 5˚C, so that the sup ernatant 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 p roven rams was p ooled, mixed and kept in a water bath at 37 ˚C. Semen was diluted 1:10 (vol. /vol.) to different diluters, then gradually cooled by p utting the tube of diluted semen in about 500 ml of 37 ˚C water containers and p laced in a 5 ˚C refrigerator for 2 hours t o reduce cold shock [24], then st ored at 5 ˚C for 72 h. Progressive motility was estimated subjectively after dilution(0h), and at 24, 48, 72 h of st orage, by diluting a drop of semen with 0.9 % sodium chlor ide solution in a warm slide, mounting it with cover slip and examining it under a microscop e at X 400 magnification, using a 100-p oint scale for linear movement [25]. The data were exp ressed in p ercentage of total cells. M otility estimations were p erformed from 5 different fields in each sample by the same p erson throughout the st udy ; the mean value averaged from 5 successive estimations was used as t he final motility score. The exp eriment was replicated 2 times with egg y olk-tris (EYT) diluter and 2 times with y olk- glucose-citrate diluter. The Statistical Analysis Syst em [26] general linear model was used to analyze the data. Differences among treatment means were compared for st atistical si gnificance, us ing Duncan's multiple range test [27]. Results Progressive motility increased si gnificantly (p < 0.01) in levels of licorice extract 1, 5, 10, 50 and 100 µg / ml in both diluters, during all st orage p eriods. The means of p rogressive motility were 72.5 ± 1.02 %, 72.08 ± 1.05, 70.90 ± 2.05 % and 66.25 ± 3.15 % resp ectively, comp ared to the control (0) 61.45 ± 16.2 %. (fig1). Levels 1, 5 and 10 µ g /ml were sup erior (p < 0.01) to levels 50 and 100 µg /ml (fig1). Diluter typ e had a significant effect (p < 0.01) on sp erm motility . Overall the p ercenta ge of motile sp erm in EYT diluter (66.48 ± 1.21 %) was higher than that in y olk-glucose citrate diluter (64.37± 1.44 %). Sp erm motility tended to decline si gnificantly (p < 0.01) as the len gth of st orage p eriod increased. The means of p rogressive 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, resp ectively. Discussion Although the hist ory of licorice is as old as the history of medicine and of confection [28] and references to licorice d ate back to approximately 2500 B.C on Assy rian clay tablets and Egy p tian p apy ri [29], this study , to the best of our knowledge, is the first indication for a p ositive effect of licorice (Gl ycyrrhiza glabra L.) on sp erm motility of chilled st ored ram semen. Sp erm motility is regarded as a man ifest ation of sp erm functional comp etence [30] and licorice extract imp roved (p < 0.01) p rogressive motility esp ecially in low levels (1, 5, 10 µg /ml diluter). Progressive motility is the most imp ortant individual quality test, because fertility is high ly correlated with number of motile sp erm inseminated [24]. IBN AL- HAITHAM J. FOR PURE & APPL. S CI. VOL.23 (1) 2010 The precise role of the comp onents of diluters (sugars, p roteins, a range of additives, etc.) in p reserving the inte grity and the fertilizing p otential of sp ermatozoa is st ill far from being understood, and the comp osition of the diluents has be en imp roved by more or less emp irical st udies [31,32,33]. Licorice may be considered as a versatile additive. First , an imp ortant reason for the decrease in motility during the storage of semen is the formation of lip id p eroxides from oxy gen radicals [34]. The sp erm p lasma membran e contains a high amount of unsaturated fatty acids and is therefore p articularly suscep tible to p eroxidative d amage [35]. The lip id p eroxidation destroy s the structure of the lip id matrix in the membranes of sp ermatoz oa, and it is associated with a loss of motility and membran e inte grity [36]. Semen contains app reciable amounts of antioxidants that balance lip id p eroxidation and p revent excessive p eroxide formation [37] but t he endogenous antioxidative capacity of semen may be insufficient during st orage or dilution [38]. The addition of antioxidants is well known to imp rove the viability and motility of liqu id st orage or cryop reserved ram sp erm cells [19, 39]. Licorice has p owerful antioxidant activity , it is contains seven antioxidant comp ounds, four isoflavans, t wo chalcon es and an isoflavone [4], and these comp ounds were shown to be effective to p rotect biolo gical sy stems against various oxidative st resses [40] via a mechanism involv ing scaven ging of free r adicals [41]. Second, licorice contains many sugars such as fructose and sucrose [11]. Fructose is one of the p rinciple ener gy substrate for sp ermatozoa [24] and an activator factor of mammal sp ermatoz oa [42], sucrose are reported to be effective in st abilizing sp erm membrane bilay ers during st orage [43]. Third, Licorice has antimicrobial activity , and the antimicrobial agents are routinely added to semen diluters to control many semen organisms which are p athogenic and compete with sp erm for nutrients and p roduce metabolic by -p roducts that have an adverse effect on livability of the sp erm [24]. The decrease in sp erm motility at high levels of licorice extract in this st udy (50 and 100 µg /ml) is p ossibly caused by high osmolality ; hyp er osmotic environment can inhibit sp erm motility [44]. Furt her researches are need ed to design an isotonic diluter with high lev el of licorice extract. Overall p rogressive motility was better in EYT. Different diluents for semen have been used to imp rove fertility rate. EYT extender was r ecommended by many researchers and commonly used for artificial insemination [23, 45]. Over st orage p eriod, sp erm motility declined si gn ificantly (p < 0.01) p robably due to the accumu lation of the toxic p roducts of sp erms metabolism [38]. The study findings may contribute to the recent attemp ts to design defined semen diluter and move away from animal-based cryop rotectants, which may p ose hy gienic r isks and are difficult to st andardize [46]. 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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 p)قیم الحركة الفردیة معنویا ازدادت. البیض مخففة والمضاف (0.01 > 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 p) المخفف معنویا نستنج من نتائج هذه . مل في قیم الحركة الفردیة / مایكوغرام 100و 50ن یستویعلى الم (0.01 > ظ مدلكباش خالل لحیامن االمخفف قد حسن من قیم الحركة الفردیة إلىمستخلص عرق السوس إضافةالدراسة ان ة الحف . ˚م 5المبرد بدرجة