AACL BIOFLUX Aquaculture, Aquarium, Conservation & Legislation International Journal of the Bioflux Society

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1 AACL BIOFLUX Aquculture, Aqurium, Conservtion & Legisltion Interntionl Journl of the Bioflux Society Effect of vitmin C nd highly unsturted ftty cids on sperm qulity of goldfish (Crssius urtus) Zein Hnee Kshni, Mohmmd R. Imnpoor, Ali Shni, nd Seed Gorgin Deprtment of Fishery, Gorgn University of Agriculturl Sciences nd Nturl Resources, Gorgn, Irn. Corresponding uthor: Z. Hnee Kshni, Z.h.kshni@gmil.com Astrct. The im of the work ws to determine the effects of three different doses of scoric cid (,, mg kg diet 1 ) nd highly unsturted ftty cid (). They were evluted on sperm qulity of goldfish. The results of the study shown tht vitmin C nd hve no significnt (P>.5) effect on sperm concentrtion, spermtocrit nd sperm motility durtion. Percentge motility of sperm in fish fed with diet C ws significntly higher thn other groups (P<.5) nd there were no significnt differences etween other groups. Key Words: vitmin C, sperm qulity,, goldfish. چکیده ھدف از این کار تعیین اثرات سھ دوز مختلف از اسید اسکوربیک (..) و اسیدھای چرب غیر اشباع بود. این مواد روی کیفیت اسپرم گل دفیش ارزی ابی شدند. نتایج این مطالعھ نشان داد كھ ویتامین C و تاثیر معني داري روي تراكم اسپرم اسپرماتوكریت و طول دوره تحرك اسپرم ندارند (/5< P). درصد اسپرم متحرك در ماھیان تغذیھ شده با جیره C بطور معني داري بالاتر از سایر تیمارھا بود (/5>P) و تفاوت معني داري بین سایر گروه ھا مشاھده نشد. کلمات کلیدی : ویتامین C کیفیت اسپرم اسیدھای چزب غیر اشباع و گلدفیش. : Introduction. There re severl gents ffecting gmete qulity in fish such s environmentl conditions nd genetic fctors. Nutrition nd content of feed (ftty cids, mino cids, minerls nd vitmins) re environmentl cues tht influence reproduction in fish (Bromge 1995; Izquierdo et l 1; Mehrd & Sudgr ). Acid scoric (AA) lso plys criticl role on gmete qulity of fish like other vertertes (Ciereszko & Drowski ). Long term feeding, using diets without AA resulted in decreses of sperm concentrtion, motility nd fertilizing ility, nd n increse in sperm lipid peroxidtion vlue (Ciereszko & Drowski 1995; Ciereszko et l 199; Drowski & Ciereszko 199; Liu et l 1997). Positive effect of AA on sperm qulity of men ws demonstrted (Dwson et l 199). Antioxidnts such s AA protect germ cells ginst to DNA dmge nd oxidtion seminl plsm proteins with rective oxygen rdicls (Frg et l 1991; Liu et l 1995). Since sperm ftty cid composition depends on the essentil ftty cid content of rood stock diet in species such s rinow trout (Oncorhynchus mykiss (Wlum, 179)) (Wtne et l 1984; Le et l 1993) nd it is possile tht sperm motility nd in turn fertiliztion would e ffected. Prticulrly in slmonids, where cryopreservtion of sperm is currently utilized, sperm ftty cid composition could e fctor tht determines the memrne integrity fter freezethwing. However, Le et l (1993) did not find ny effect of dietry ftty cids n3 nd polyunsturted ftty cids n on sperm freeze thw fertilizing ility, wheres low memrne cholesterol phospholipids rtios were correlted with etter sperm freezing resistnce (Le & Misse 199). 39

2 Mteril nd Method. A numer of goldfish (Crssius urtus (Linneus, 1758)), verge.9±.1 g initil weight, from lrvl stge to roodstock were used s n experimentl fish nd the feeding tril ws conducted in the Aquculture Lortory, Aquculture Reserch Center of Fisheries Deprtment in Gorgn University of Agriculturl Sciences nd Nturl Resources. Fish were fed with diets contining different level of vitmin C nd highly unsturted ftty cid: C, C, C, C, C, C. The generl composition of ech experimentl diet is given in Tle 1. Ingredient (g g diet 1 ) nd chemicl proximte composition (% Dm) of experimentl diets Ingredient Corn mel Fish mel Soyen mel Bred flour Rice rn Fish oil Soyen oil Minerl mixture Vitmin mixture Lysine Metionin Anti fungi Dm: dry mtter. C C C C C Tle 1 C Minerl mixture (g kg 1 diet): C(PO 4H ) H O(3), CCO 3 (.5), KCl (.5), NCl (4), MnSO 4 H O (.), FeSO 4 7H O (1.5), MgSO 4 (4.), KI (.), CuSO 4 5H O (.5), ZnSO 4 7H O (.), CoSO 4 7H O (.5), N SeO 3 (.18 ), Al (SO 4)3 18H O (1 ). Vitmin mixture (mg kg 1 diet): Thimine (4), Rioflvin (), Pyridoxine (3), Pnthotenic cid (15), Nicin (5), Folic cid (15), Inositol (), Choline (5), Biotin (3), Cynocolmin (.5), Vitmin A (1), Mendion (5). Fish were fed 3 times dy with experimentl diets for 1 yer. After tht mle goldfish were selected rndomly from ech tretment (totlly 3 mles) stripped of ll milt in dry nd cool glss vil. Before stripping, the nl regions of the fish were clened y dry towel. The nl nd seminl ducts were emptied to void contmintion of smples with ure nd feces. All sperm mnipultions were performed on ice. Semen smpled for qulity estimtes ws used within 8 h fter stripping. In order to estimte spermtozo concentrtion, milt ws diluted with sline solution nd counted. Micro hemtocrit tues (75mm length, internl dimeter) were used for determintion of spermtocrit. The triplicte tues for ech smple were filled up with milt nd one side of the tues ws covered with glss putty nd plster. They were centrifuged 3g for 8 min nd mesured using hemtocrit reder. Sperm ws diluted using sline solution rtio of 1:. Ten microliters of diluted semen were plced in ovine serum lumincoted microscope slide. The percent motility of ech sperm smple ws estimted using light microscopy t x4 mgnifiction immeditely fter ddition of ml of wter used s n ctivting solution (Figure 1). Motility ws determined s the percentge of sperm ctively moving forwrd. Ten microlitres of diluted semen ws ctivted under x mgnifiction to determine the motility durtion. The time intervl from 5 s fter ctivtion of sperm to when ll the sperm stopped ws considered to e the motility durtion. The smples of diets were dried to constnt weight t 5 C to determine the dry mtter content. Protein ws determined 33

3 y mesuring nitrogen (N.5) using the Kjeldhl method; lipid y ether extrction using Soxhlet (AOAC 1995). Figure 1. Stereomicroscopic imges nlyze y specil soft. All dt were sujected to onewy nlysis of vrince. The significnce of difference etween mens ws determined using Duncn s multiple rnge test (P<.5) using SPSS for WINDOWS (Version 1.). Vlues re expressed s mens ± SE. Results nd Discussion. Sperm concentrtion, spermtocrit vlues, motility durtion were not significntly different etween groups (P>.5). There were significnt differences in the percent motility of sperm (P<.5). Highest percent motility of sperm ws detected in fish fed with the diet with C. There ws no significnt difference etween groups (Figure ). The present study show tht ddition of vitmin C nd hve no effect on sperm concentrtion, spermtocrit vlues nd motility durtion of goldfish mles. Goldfish mles given mg kg diet 1 AA supplemented diet for 1 months shown the est percent motility of sperm mong C groups. Although the AA mechnism of how to effect sperm numer in nimls is not known clerly, there re severl reserches tht reported positive effect of AA on sperm concentrtion in humn nd nimls. Cnyurt & Akhn (8) suggested tht dditionl AA is essentil for mle rinow trout rood stock. AA positively ffected some prmeters of sperm qulity in rinow trout, such s sperm concentrtion, motility nd spermtocrit vlues. They showed tht motility incresed with AA supplementtion, ut our study reveled tht AA hs no effect on this prmeter. In this study, hs no effect on sperm qulity prmeters in tretment groups except percent motility of sperm. Addition with mg kg diet 1 incresed percent motility of sperm. Since sperm ftty cid composition depends on the essentil ftty cid content of rood stock diet in species such s rinow trout (Wtne et l 1984; Le et l 1993) it is possile tht sperm motility nd in turn fertiliztion would e ffected y content. 331

4 sperm concentrtion sperm concentrtion C C CHUF A _CHUF A _C CHUF A C HU FA C HU FA CHU FA _C _C C HU FA percent motlity of sperm sperm motility durtion C C C _C _C C C1 HU FA C1 C _C _C C1 Fig.. A Spermtocrit (%) in different tretment groups of goldfish; B sperm concentrtion in different tretment groups of goldfish; C percentge motility of sperm (%) in different tretment groups of goldfish; D sperm motility durtion (s) in different tretment groups of goldfish. Conclusions. The ntioxidnt cpcity of Lscoric cid (LAA) effects sperm concentrtion, motility nd fertilizing ility positive protective role of LAA on mle germ cells re known, ut lter reserches my e designed to explin how LAA increse sperm concentrtion in mle goldfish. Generlly LAA increse the mturtion of germ cells which led to increse milt qulity such s sperm concentrtion, motility durtion nd fertility. In conclusion, the ntioxidnt cpcity of Lscoric cid positively effects sperm concentrtion, motility nd fertilizing ility. Results of this study showed tht dditionl AA supplementtion is useful for improving percent motility of sperm. Protective role of AA on mle germ cells re known, ut lter reserches my e designed to explin how AA increse sperm viility in mle goldfish. Acknowledgements. The uthors would like to thnk Gorgn University of Agriculturl Sciences nd Nturl Resources for providing the necessry fcilities for the study. References Assocition of Officil Anlyticl Chemists (AOAC), th Edition, A.O.A.C., Wshington DC, 1:

5 Bromge N. R., 1995 Brood stock mngement nd seed qulitygenerl considertions. In: Broodstock mngement nd egg nd lrvl qulity. Bromge N. R., Roerts R. J. (eds.), Blckwell Science Ltd., Oxford, pp. 14. Cnyurt M. A., Akhn S., 8 Effect of scoric cid supplementtion on sperm qulity of rinow trout (Onchorynchus mykiss). Turkish Journl of Fisheries nd Aqutic Sciences 8: Ciereszko A., Drowski K., 1995 Sperm qulity nd scoric cid concentrtion in rinow trout semen re ffected y dietry vitmin C: n crossseson study. Biology Reproduction 5(5): Ciereszko A., Drowski K., Effect of scoric cid supplement in vitro on rinow trout sperm viility. Biologicl Science 8:18. Ciereszko A., Liu L., Drowski K., 199 Effect of seson nd dietry scoric cid on some iochemicl chrcteristics of rinow trout (Oncohrynchus mykiss) semen. Fish Physiology nd Biochemistry 15(1):1. Drowski K., Ciereszko A., 199 Ascoric cid protects ginst mle infertility in teleost fish. Biomedicl nd Life Science 5():97. Dwson E. B., Hrris W. A., Tetter M. C., Powell L. C., 199 Effect of scoric cid supplementtion on sperm qulity of smokers. Fertility nd Sterility 58(5): Frg C. G., Motchnick P. A., Shigeng M. K., Helock H. J., Jco R. A., Ames B. N., 1991 Ascoric cid protect ginst endogenous oxidtive DNA dmge to humn sperm. PNAS 88(4):131. Izquierdo M. S., FernndezPlcios H., Tcon A. G. J., 1 Effect of roodstock nutrition on reproductive performnce of fish. Aquculture 197(14):54. Le C., Misse G., 199 Influence of rinow trout therml cclimtion on sperm cryopreservtion: reltion to chnge in the lipid composition of the plsm memrne. Aquculture 145(14): Le C., Loir M., Kushik S., Misse G., 1993 The influence of oth rering nd dietry lipid origin on ftty cid composition of spermtozon polr lipids in rinow trout (Oncorrhynchus mykiss). Effect on sperm cryopreservtion tolernce. Fish Nutrition in Prctice, Birritz (Frnce), June 4 7, EdINRA, Pris 1993 (Les Colloques, no. 1), pp Liu L., Drowski K., Ciereszko A., 1995 Protective effect of seminl plsm proteins on the degrdtion of scoric cid. Mol Cell Biochem 148(1):59. Liu L., Ciereszko A., Drowski K., 1997 Dietry scoryl monophospht depress lipid peroxidtion in rinow trout spermtozo. Journl of Aqutic Animl Helth 9(4):4957. Mehrd B., Sudgr M., The effect of vitmin C on growth fctors, survivl, reproduction nd sex rtio in guppy (Poecili reticult). AACL Bioflux 3(3): Wtne T., Tkeuchi T., Sito M., Nishimur K., 1984 Effect of low protein high clorie or essentil ftty cid deficiency diet on reproduction of rinow trout. Nippon Suisn Gkkishi 5(7): Received: 11 Decemer. Accepted: 1 Mrch 11. Pulished online: 1 April 11. Authors: Zein Hnee Kshni, Deprtment of Fishery, Gorgn University of Agriculturl Sciences nd Nturl Resources, Irn, Golestn, Gorgn, Shhid Beheshti Avenue, Postl code: , emil: z.h.kshni@gmil.com Mohmmd Rez Imnpoor, Deprtment of Fishery, Gorgn University of Agriculturl Sciences nd Nturl Resources, Irn, Golestn, Gorgn, Shhid Beheshti Avenue, Postl code: , emil: mrimnpoor@yhoo.com Ali Shni, Deprtment of Fishery, Gorgn University of Agriculturl Sciences nd Nturl Resources, Irn, Golestn, Gorgn, Shhid Beheshti Avenue, Postl code: , emil: li_shni@yhoo.com Seed Gorgin, Deprtment of Fishery, Gorgn University of Agriculturl Sciences nd Nturl Resources, Irn, Golestn, Gorgn, Shhid Beheshti Avenue, Postl code: , emil: s_gorgin@gmil.com How to cite this rticle: Hnee Kshni Z., Imnpoor M. R., Shni A., Gorgin S., 11 Effect of vitmin C nd highly unsturted ftty cids on sperm qulity of goldfish (Crssius urtus). AACL Bioflux 4(3):