Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 12 EFFECT OF VITAMIN C INJECTION ON SEMEN QUALITY OF AWASSI RAMS. H.K.J.Al-saab* * Department of Animal Resource- College of Agriculture - University of Baghdad- Iraq Dr.hazim_alsaab@yahoo.com. ABSTRACT This study was conducted in the Animal Farm, College of Agriculture, University of Baghdad, from march to may 2013, to clarify the effect of subcutaneous vitamin C injection on semen characteristics of Awassi rams. Six rams (1.5-2 years old) were randomly divided into three groups of 2 rams. Rams in T1and T2 were subcutaneously injected, every other day with dose of 50 and100 mg/kg body weight respectively ,for 45 days while control group in T0 received normal saline. The results showed a significant increase (P<0.05) in the The sperm concentration of T1 as compared with control group, while there were no significant differences among treated groups(T1 and T2). Group 2 showed a significant (P<0.05) increases in mass activity, individual motility and live sperm percentage. There was a significant decrease in individual motility and percentage of live sperm in T2 as compared to T1 and control group. In conclusion, despite of the significant improvement of some semen trail as in T2 group, the lower dose of vit. C(less than 50 mg/kg of body weight) was recommended represented the skin tearing(T2group) as well as the redness and sensitive skin in T1 group. Key words:Vitamin C,Semen,Rams. INTRODUCTION Vitamin C, is a water soluble vitamin also known asscorbate or ascorbic acid(AA), synthesize from glucose, by the active enzyme L-gulonolactone oxidase (Linster& Van Schaftingen, 2007), L- ascorbic acid and L- dehydroascorbic acid are physiologically active forms of vitamin C (Thurnham, 2000). Vitamin C has been associated with fertility, and it may have It has also been shown to be important for reproduction (Akmal et al,2006). Deficient ascorbate in man have been associated with low sperm counts, agglutination,increased number of abnormal sperm, and reduced fertility, (Wilson,1954; Harris et al., 1979; Dawson et al.,1990). The (AA) level of ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Received for publication Feb. 11 , 2014 . Accepted for publication Dec. 7 , 2014 . Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 13 seminal plasma were found to be positively correlated with sperm concentration, sperm motility, normal sperm morphology. and closely related to male fertility(Ko et al 2012). There is beneficial effect of subcutaneous injections of (AA)to sub-fertile bulls (Phillips et al., 1940) and stallions (Ralston et al., 1988)in addition to increased its plasma and seminal concentrations (Fazeli et al., 2010). In rabbits, supplementation with vitamins C and E reduced the production of free radicals and improved semen quality (total sperm output, sperm concentration and ejaculate volume) (Yousef et al. 2003). The small beneficial effect of feeding of (AA) reported in rams (Asghari, 1999) and goats (Heidari, 2002) might be due to denaturing of this vitamin in the rumen(Hidiroglou et al., 1997).Sonmez and Demirci (2003) reported that intramuscular injection of 20 mg (AA)per kg live weight for 30 days increased semen volume, sperm concentration and motility of Rams,So Vitamin C is a physiological additives because it help the normal development of physiological functions of deferent activity in the body(Peris and Calafat, 2005). So this experiment was done to evaluate the subcutaneous injection of Vit. C on some physiological activity of semen of Awassi rams. MATERIALS AND METHODS Six mature Awassi rams (1.5-2 years of age) weighing 40-55kg were used in this study, The study was carried out from march to may 2013. Animals were housed at the Animal Farm of the College of Agriculture, University of Baghdad,. All rams were in good health. They were maintained in identical nutritional and managerial condition throughout the period of study. The rams were randomly divided into three groups (2 rams each) and received subcutaneous injections of either 0, 50 or 100 mg vitamin C per kg live weight once every two day for 45 days. The experiment was started on march 16, and data were collected from march 31, to 30 may, 2013. Vitamin C (L(+)- ascorbic acid, Carlo Erba-Reagenti Vale De Reuil) was dissolved in distilled water and filtered through a 25 μm filter before injection. The control animals were injected with normal saline. A total number of 48 ejaculates were collected from the rams using an electro ejaculator twice weekly. Ejaculates were evaluated and accepted, if the following criteria were met: volume varying between 0.4-1.0 ml; sperm concentration of 1-2 x 10 9 sperm/ml;the motile sperms percentage higher than 70% and less than 12% dead sperm in total. Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 14 Semen analysis Semen collection 15 days after the first injection.Fresh semen samples were kept in a water bath at 35°C, and seminal parameters (volume, pH,sperm concentration, individual motility, percent of live sperm, percent of sperm showing morphological abnormalities) were evaluated within 15 min (Evans and Maxwell, 1989). Semen volume was determined by direct reading the graduated tubes .Semen pH was measured immediately using a digital pH meter (pH 211Microprocessor, Hanna, Italy).Mass motility was estimated immediately after semen collection using a microscope (Olympus,Japan) (x100 magnification), fitted with a warm stage at 37°C (Zamfirescu & Sonea, 2004). Sperm concentration was determined by Neubaur hemocytameter,diluting a small drop of the semen with 2% eosin solution (1:200), with the sperm counted in five squares of one chamber (Smith and Mayer 1955). The percentages of individual motility were determined microscopically (×400) by using a score ranging from 0 to 100%( Chemineau et al, 1991). The percentages of live sperm were determined by observing 200 spermatozoa, after eosin-nigrosin staining,using a light microscope at ×400 magnification (Evans and Maxwell, 1989).the final score being the mean of three successive estimations Statistical Analysis Statistical analysis was performed by using SAS program (SAS,2010). The significant level was set at P≤0.05. Duncan’s multiple range test(Duncan,1955)was used for comparisons among means.The statical model used was: Yij=µ+τi+εij Where: Yij…………………..Observed value µ…………………….Overall mean τi……………………The effect of the treatment(i=1,2,3) εij……………………Error RESULTS AND DISCUSSION In this study, the effect of vitamin C administration as antioxidant on some semen characteristics in Awassi rams was investigated. The results of this experiment were listed in Tables 1and 2 confirm that there was a significant decrease (P<0.05) in the ejaculate volume in both treated groups, while there were no significant differences showed among treated groups . There were no significant differences in the pH of seminal plasma among all groups. Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 15 Table 1: Effects of vitamin C injection on ejaculate volume , pH and sperm Concentration of Awassi rams. T0=control group , T1= subcutaneous injections of 50 mg/kg Vit. C , T2= subcutaneous injections of 100 mg/kg Vit. C Different capital letter in the column means significantly different ( p ≤ 0.05 ). Table 2: Effects of vitamin C injection on mass activity, individual motility and sperm viability of Awassi rams. T0=Control group , T1= subcutaneous injections of 50 mg/kg Vit. C , T2= subcutaneous injections of 100 mg/kg Vit. C Different capital letter in the column means significantly different ( p ≤ 0.05 ). The sperm concentration were significantly increased (P<0.05) in rams of T1 compared to control group, Similarly, Sonmez. and Demirci (2003 )reported that intramuscular injection of 20 mg Vit.C / kg body weight for 30 days increased sperm concentration in rams Yousef et al.(2003) reported that adding ascorbic acid to drinking water for 12 weeks increased sperm concentration of male rabbits..while there were no significant differences showed among T2 and control group and between the two treatment groups(2 and 3). Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 16 The results of sperm motility (Table 2) demonstrates that rams in T1 showed a significant (P<0.05) increase ,which agreed with Sonmez and Demirci (2003) when they found that the intramuscular injection of ascorbic acid for 30 days increased the semen quality of rams. However short-term administration of vitamin C did not affect semen parameters of rams (Sonmez and Tanyildizi,2008). Fazeli et al.(2010) found the importance of subcutaneous vitamin C injections for 90 days in increased of about 15% in sperm motility of buck. Our observations indicating that the vitamin C was able to improve some sperm parameters. Furthermore, the motility of sperm cells would be preserved by the binding of antioxidant to endoperoxides (Luvoni ,2006). The mechanism of Vitamin C effect may due to inhibition of cell impair by binding to the free radical and neutralizing its unpaired electron mediated by a tocopheryl-quinone' formation (Schuh, et al. 2004). and improve ram sperm parameter after the addition of vitamins C and E to semen diluents (Thuwanuta et al.2011). However, in this study higher doses of Vit. C 100mg/kg (T2 )showed significant decrease in the individual motility and percentages of live sperm compared to rams of T1 and control groups , as well as it cause skin tearing, and this may be due to the harmful effect of high vitamin C concentrations to sperm motility ( Beconi et al. 1993) ,and may have damaging pro-oxidant effects which actually reduce sperm motility (Abel et al.,1983). REFERENCES Abel, B. J., G. Carswell and R. A. Elton.1983. Randomised trial of clomiphene citrate treatment and vitamin C for male infertility. Br. J. Urol., 54, 780– 784. Akmal, M., J. Q. Qadri, N.S. Al-Waili, S. Thangal, A. Haq and K.Y. Saloom .2006. Improvement in human semen quality after oral supplementation of vitamin C.J Med Food.;9(3):440-2. Asghari, S. R. 1999. Testicular and seminal characteristics of Ghezel and Mehraban rams as affected by season and vitamin C supplementation. MSc Thesis, Shiraz University, Iran. P: 92 (In Persian with English abst.). Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 17 Beconi M. T., C. R. Francia, N. G. Mora and M. A. Affranchino .1993.Effect of natural antioxidants in frozen bovine semen preservation. Theriogenology; 40: 841-851. Chemineau, P., Y. Cagnie, Y. Guerin, P. Orgeur and J. C. Vallet . 1991. In: Training Manual on Artificial Insemination in Sheep and Goats.FAO Anim. Prod Hlth. Paper 82, Rome,: 115-161. Dawson, E. B., W. A. Harris and L. C. Powell 1990. Relationship between ascorbic acid and male fertility. World Rev. Nutr. Diet., 62:1-26. Duncan, D. B.1955.Multiple range and multiple F tests. Biometrics 11:1-42. Evans, G. and W. M. S. Maxwell . 1989. Salamon’s Artificial Insemination of Sheep and Goats.Sydney, NSW, Australia, University Press. PP:194. Fazeli, P., M. J. Zamiri, A. Farshad and B. Khalili. 2010. Effects of vitamin C on testicular and seminal characteristics of Markhoz goats. Iranian Journal of Veterinary Research, Shiraz University, Vol. 11, No. 3, Ser. No. 32.p 267-272.Short paper. Harris, W. A., T .E. Harden and E. B. Dawson.1979. Apparent effects of ascorbic acid medication on semen metal levels. Fertil. Steril., 32: 455- 459. Heidari, A. H. 2002. Testicular and seminal characteristics of Rayini goats as affected by season and vitamin C supplementation. MSc Thesis, University of Sistan and Baluchistan,Iran. P: 42 (In Persian with English abst.). Hidiroglou, M., T. R. Batra and X. Zhao. 1997.Comparison of vitamin C bioavailability after multiple or single oral dosing of different formulation in sheep. Reprod. Nutr. Dev., 37:443-448. Ko E. Y., S. Edmund and J. R. Sabanegh. 2012. The role of over-the-counter supplements for the treatment of male Infertility -fact or fiction? J. Androl. ; 33:292–308. Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 18 Linster, C. L. and E.Van Schaftingen. 2007. Vitamin C. biosynthesis, recycling and degradation in mammals. FEBS J 274: 1-22. Luvoni, G. C. 2006.Gamete cryopreservation in the domestic cat. Theriogenology; 66: 101-111. Peris, S. and F. Calafat.2005.Acidification and other physiological additives. industrial tecnicapecuaria (ITPSA), Avda. Roma. Phillips, P. H., H. A. Lardy, E.E. Heizer and I.W. Rupel.1940. Sperm stimulation in the bull through the subcutaneous administration of ascorbic acid. J. Dairy Sci., 23: 873-878. Ralston, S. L., S. Barbacini, E. L. Squires and C. F. Nockels.1988.Ascorbic acid supplementation in stallions. J. Equine Vet.Sci., 8: 290-293. SAS. 2004. SAS / STAT User's Guide for Personal Computers. SAS Institute, .Inc. Cary, N. C. USA. Schuh, K., E. J. Cartwright ,E. Jankevics, K. Bundschu, and J.Liebermann .2004 .Plasma membrane Ca 2+ ATPase 4 is required for sperm motility and male fertility, J Biol Chem, 279: 28220–28226. Smith, J.T., and D.T. Mayer .1955. Evalution of sperm concentration by the haemocytometer method.comparison of four counting fluid.Fert.Steril.,6:271-275. Sonmez, M. and E. Demirci.2003. The effect of intramuscular vitamin C administration on semen quality in rams. J. Firat Univ. Health Vet. Sci., 17: 195-201. Thurnham, D. I. 2000. Water-soluble vitamins (Vitamin C and B Vitamins, Thiamin, Riboflavin and Niacin). In: “Human Nutrition and Dietetics”. Garrow, J.S., W.P.T. James, A. Ralph (Eds.). Churchill. Livingston Publishers, Edinburgh London, 249-257. Thuwanuta, P., K. Chatdarongb and A.S. Bergqvista.2011. The effects of antioxidants on semen traits and in vitro fertil-izing ability of sperm Al-saab Diyala Agricultural Sciences Journal, 7 ( 2 ) 12 – 19 ,2015 19 from flat-headed cat (Prionailurus planiceps). Theriogenology; 76: 115- 125. Wilson, L. 1954. Sperm agglutination in human serum and blood. Proceedings of the Society for Experimental Biology and Medicine. 85:652-655. Yousef , M. I., G .A. Abdullah and K.I. Kamel. 2003. Effect of ascorbic acid and vitamin E supplementation on semen quality and biochemical parameters of male rabbits. Anim Reprod Sci.;76(1-2):99-111. Zamfirescu S, Şonea A, 2004, Biotehnologii de reproducţie la rumegătoarele mici, Ed Ex Ponto Constanţa, pag 170-180. . على نوعیة السائل المنوي للكباش العواسي C حقن فیتامین تأثیر حازم كسار جاسر الصعب .Dr.hazim_alsaab@yahoo.com جامعة بغداد–كلیة الزراعة - قسم الثروة الحیوانیة -مدرس* مستخلصال بو غریب للفترة ألكلیة الزراعة/ جامعة بغداد في في الحقل الحیواني التابعھذه الدراسة أجریت لسائل على بعض صفات ا Cالحقن تحت الجلد لفیتامین تأثیرلمعرفة ، 2013مایس ولغایة آذار من سنة وقسمت الى ثالث مجامیع وبواقع كبشین 2-1.5ستة كباش بعمر تالمنوي للكباش العواسي. استخدم /كغم من وزن Cملغم فیتامین 100و 50كباش المجموعتین الثانیة والثالثة حقنت بـ لكل مجموعة. .لمحلول الفسیولوجيامجموعة سیطرة وحقنت ب األولىالمجموعة ، ایوم 45 الجسم بین یوم وآخر لمدة نتائج ھذه التجربة زیادة اظھرت تم جمع السائل المنوي من الكباش باستخدام التحفیز الكھربائي . فیما لم تظھر الثانیة مقارنة بمجموعة السیطرة ، لكباش المجموعة نطففي تركیز ال (P<0.05)معنویة وعة الثانیة والثالثة. المجموعة الثانیة فروق معنویة بین المجموعة الثالثة والسیطرة وكذلك بین المجم كما لوحظ حصول الحیة. نطفونسبة ال نطفاظھرت زیادة معنویة في الحركة الجماعیة والفردیة لل الحیة لكباش المجموعة الثالثة مقارنة بالمجموعة نطفانخفاض معنوي في الحركة الفردیة ونسب ال الثانیة ومجموعة السیطرة. للمجموعة على الرغم من التحسن المعنوي لبعض صفات السائل المنوي نھ ویمكن االستنتاج با تاثیرات البسبب /كغم من وزن الجسم Cفیتامین ملغم 50باستخدام تراكیز اقل من وصياال اننا ن ,الثانیة واحمراروتحسس )الجلد تكتھ( المجموعة الثالثة فيالتراكیز العالیة السلبیة التي لوحظت عند استخدام .الثانیة لدى المجموعة (تھرش )الجلد . الكباش، السائل المنوي ، Cفیتامین الكلمات المفتاحیة: