Vahid Taghizadeh, Mohammad Reza Imanpoor and Ali Sadeghi

Transcription

1 World Journl of Fish nd Mrine Sienes (): , 13 ISSN IDOSI Pulitions, 13 DOI: /idosi.wjfms Effet of Extenrs nd Different Conentrtions of Methnol on Motility Prmeters of Goldfish (Crssius urtus gielio) Spermtozo fter ShortTerm Storge Vhid Tghizh, Mohmmd Rez Imnpoor nd Ali Sghi Deprtment of Fisheries, Fulty of Fisheries nd Environmentl Sienes, Gorgn University of Agriulturl Sienes nd Nturl Resoures, Irn Astrt: Semen smples were olleted from eight mles of Goldfish (Crssius urtus gielio) nd ryopreserved using four extenrs (Goldfish sperm extenrs; GFSE 1 GFSE 4) supplemented with Methnol (MeOH) t onentrtion of %, 10% nd %. Semen ws diluted with rtio of 1:1 with extenrs nd frozen in liquid nitrogen vpor. Frozen sperms fter 2 nd dys were exlud from freezing. Experiment showed the highest motility durtion nd the most motility perentge of postthwed sperms fter 2 dys for sperm frozen t extenr GFSE 2 with the onentrtion of MeOH 10%(31.66±8.02 s nd 16.66±3.1%; P<0.0), s well s the upmost moility nd the motility durtion of postthwed sperms fter dys ws oserved for sperm frozen t extenr GFSE 2 with the onentrtion of MeOH10% (22.66±3.78 s nd 13±2.64%; P<0.0). Key words: Goldfish Sperm Methnol Motility Durtion Motility Perentge INTRODUCTION out during the spwning sesons euse the qulity nd quntity of spermtozo is highest t this time. In Se The Goldfish (Crssius urtus gielio) is ss, for exmple, the onentrtion of spermtozo freshwter fish in the Cyprini fmily tht is similr to resed s the spwning seson progressed [3]. Common rp (Cyprinus rpio) in iologil nd Similrly, Suquet et l. [4] reported lower motility rtes, nutritionl spets [1]. The Goldfish ssoited with fertiliztion rtes nd redued shortterm storge the ulture nd eliefs of people round the world nd pity t the end of the reprodutive period of the fish. is lso n importnt fish spets of reserh nd Extenr omposition nd ryoprotetnt onentrtion eonomilly. It hs een estimted tht sperm from re known to ffet ryopreservtion suess [, 6]. 0 fish speies hve een suessfully Cryopreservtion of sperm is simple tehnique tht is Cryopreserved [2], however, speiesspeifi useful for speies onservtion, preservtion of optimiztions of tehnology re need. Importnt iodiversity [7], protetion of vlule reeding lines prmeters for ryopreservtion inlu types of nd s helpful tool in filitting niml reprodution. extenrs nd ryoprotetnts, the dilution rtio, the During ryopreservtion, numer of ftors my lter freezing nd thwing rtes nd type of extenr used for the physiologil sttus of spermtozo. The min ftors fertiliztion. The most effiient permeting ryoprotetnt re: ryoprotetnt used, its onentrtion, omposition for fish milt ppers to e Dimethyl Sulfoxi (DMSO) of the diluent, milt dilution rtio, equilirtion time nd nd Methnol (MeOH). The highest effiieny of rtes of freezing nd thwing [8, 9]. Cryopreservtion DMSO nd MeOH is t pproximtely 10% onentrtion. suess ws usully mesured s postthw sperm Other ryoprotetnts suh s Glyerol, Ethylene glyol, moility [10, 11] or s fertiliztion suess during primry Propnediol, Dimethyl Aetmi nd re less populr or emryo growth [12]. The ojetive of the present study hve een used with limited suess. ws to termine the most suitle extenr nd Sperm ryopreservtion is one wy to overome onentrtion of Methnol for Goldfish (Crssius the prolems ssoited with rood stok supply. urtus gielio) spermtozo dilution for Sperm olletion for ryopreservtion should e rried ryopreservtion. Corresponding Author: Vhid Tghizh, Deprtment of Fisheries, Fulty of Fisheries nd Environmentl Sienes, Gorgn University of Agriulturl Sienes nd Nturl Resoures, Gorgn, Irn. 492

2 World J. Fish & Mrine Si., (): , 13 Tle 1: The ompositions of vrious extenrs used in the experiments Type of extenrs Chemils GFSE1 GFSE2 GFSE3 GFSE4 NCl g KCl 0.46 g 0.46 g Frutose g g Surose g g Distilled wter 100mL 100mL 100mL 100mL NoH solution 21µL 16µL µl 17µL Antiioti 0.mL 0.mL 0.mL 0.mL ph GFSE: Goldfish sperm extenrs NOH solution (NOH 1 g + distilled wter 100 ml) for djusting ph. Antiioti 10,000 Unit/mL peniillin nd 10,000_g/mL streptomyin (Pen Strep from GIBCO BRL, t. no 1140) MATERIALS AND METHODS supplemented with MeOH t onentrtion of %, 10% nd %. Pooled semen ws prepred y mixing Sperm Colletion for Cryopreservtion: Semen smples equl portions of semen olleted from eight mles. were olleted from eight mles of Goldfish (Crssius Pooled semen ws then diluted 1:1 with extenrs. urtus gielio) in the Reserh Center of Aquulture of Suspensions of extend milt were drwn within 0.2ml Fzli Brdi mrtyr, Gorgn University of Agriulturl strws. Semenfreezing ws onduted in styrofom ox Sienes nd Nturl Resoures, Gorgn, Irn. filled with liquid nitrogen. Strws were pled on 4mhigh floting frme m of styrofom. Strws were seled Assessment of Sperm Qulity: Motility of sperm smples nd fter 3 min of freezing in liquid nitrogen vpor, were ws estimted unr light mirosope t 400 plunged within liquid nitrogen [14]. mgnifition immeditely fter mixing of µl of sperm with 0µL of distilled wter on mirosope sli. Sperm Mesurement Sperm Moility nd Durtion of Sperm motility nd durtion of sperm motility ws reord using Motility: Strws were thwed in wter th with softwre gdmei tv home medi v. 330 from note ook temperture of 60 C for 10 s [13]. Sperm moility nd onneted to Nikon mirosope (Optiphot2, Jpn) t durtion of sperm motility of thwed semen ws oserved 400 mgnifition tht omined with CCD olor vio fter 2 nd dy of storge in liguid N2.Postthw mer (mol SPC00P, Jpn). Sperm motility nd moility nd motility durtion ws oserved nd durtion of sperm motility were evluted from sperm with evluted y the sme opertors using monitor forwrd movement. Immotile sperm were fined s sperm onneted to mirosope. tht did not show forwrd movement fter tivtion. Vio reords were set t 30 frmes/se using vio Dt Anlysis: All vlues re shown s men±s.d. mer mounted on mirosope. Perentge of sperm Dt for perentge nd durtion of sperm motility motility ws termined during 0 1 s posttivtion. sttistil nlysis y SPSS v16.0 softwre. The effets of Motility durtion ws evluted y ounting the time from extenrs on postthw sperm motility nd durtion of sperm tivtion with distilled wter until sperm stopped sperm motility were nlyzed using onewy nlysis of moving. Anlyses were repeted three times for eh vrine (ANOVA). tretment. Only sperm smples showing 80% motility or higher were used for the experiments [13]. RESULTS Extenrs nd Sperm Cryopreservtion: Extenrs were Effets of Extenrs nd Conentrtions of MeOH on veloped from sugrs suh s Frutose nd Surose nd Qulity PostThwed Sperms fter 2 Dys: Sperm frozen ion sed hemils suh s NCl nd KCl. The extenrs t GFSE 2 with the onentrtion of MeOH 10% hd the were simplified s Goldfish sperm extenr (GFSE) highest postthw motility (16.66±3.1%) while other onsisting of vrious omponents (CCSE1 CCSE4) tretments hd verge postthw sperm motility etween shown in Tle 1. All extenrs were djusted to ph of 2.33 nd 1.66% (Tle 2). Longest durtion of postthw 7.7 with NOH nd kept t 2 C efore use. All extenrs sperm motility (31.66±8.02s) ws oserved from sperm 493

3 World J. Fish & Mrine Si., (): , 13 Tle 2: Effets of extenrs nd different onentrtions of MeOHon postthw sperm motility nd durtion of sperm motility fter 2 dys of freezing Type of extenrs Cryoprotetnt onentrtion (%) Cryoprotetnt Motility durtion (s) Motility perentge (%) 19.33± ±3.1 GFSE1 10 MeOH d 23.00± ±2.1.66± ± ± ±3.0 GFSE2 10 MeOH 31.66± ± ± ± ± ±2.00 GFSE3 10 MeOH 18.66±4.72 d 8.00± ± ±1.2 e 14.00±4.00 e 2.33±1.2 GFSE4 10 MeOH 1.33± ± ± ±2.1 Control ± ±6.18 Vlues within olumn followed y different supersript letters were signifintly different (P<0.0) Tle 3: Effets of extenrs nd different onentrtions of MeOHon postthw sperm motility nd durtion of sperm motility fter dys of freezing Type of extenrs Cryoprotetnt onentrtion (%) Cryoprotetnt Motility durtion (s) Motility perentge (%).00± ±2.08 GFSE1 10 MeOH 19.66± ± ± ± ± ±2.30 GFSE2 10 MeOH 22.66± ± ± ± ± ±1.00 GFSE3 10 MeOH 9.66± ± ± ± ± ±1.1 GFSE4 10 MeOH 7.00± ± ± ±0.7 Control ± ±6.18 Vlues within olumn followed y different supersript letters were signifintly different (P<0.0) frozen t GFSE 2 with the onentrtion of MeOH 10%. DISCUSSION Lest durtion nd the lowest moility of postthw sperm motility were oserved from sperm frozen t GFSE 4 with Most extenrs in the present study were used the onentrtion of MeOH % (14±4 s nd 2.33±1.2%; suessfully in freezing Cyprini semen. GFSE 2 with P<0.0). the onentrtion of MeOH 10% ws the most suitle omintion of extenr for ryopreservtion of Goldfish Effets of Extenrs nd Conentrtions of MeOH on sperm sed on the presene of highest postthw sperm Qulity PostThwed Sperms fter Dys: Mximum motility in omprison with other omintions. This my motility durtion nd the upmost moility of postthwed e due to synergisti effet resulting in superior sperms fter dys ws relted in the tretments with performne fter freezing. Extenrs GFSE 2 onsists of extenr GFSE 2 nd the onentrtion of MeOH 10% NCl nd surose where Surose funtioned s non (22.66±3.78 s nd 13 ±2.64%; Tle 3), While the minimum permeting ryoprotetnt tht my protet the externl durtion motility of postthwed sperms ws oserved in prt of the sperm ell [1] or stilize liposoml the tretments with extenr GFSE 4 nd the memrnes during freezing [16]. Cryopreserved sperm is onentrtion of MeOH 10% (7±2 s). Lest moility of relile proedure for the mintenne of fish speies postthw sperm motility were oserved from sperm euse of its reltive esiness for freezing nd storge. frozen t GFSE 4 with the onentrtion of MeOH % Methnol s n internl ryoprotetnt signifintly (1.66±1.1%; P<0.0). improved motility, progressive motility nd its durtion of 494

4 World J. Fish & Mrine Si., (): , 13 ryopreserved sperm. Methnol ws employed s 3. Fuvel, C., O. Svoye, C. Drenno, J. Cosson nd suessful internl ryoprotetnt in other Cypriniforms M. Suquet, Chrteristi of sperm of Hypophthlmihtys molitrix [17]; Tor khudree [18]; Tin ptive Se ss (Dientrrhus lrx) in tin [19]; Cyprinus rpio []; Dnio[21]. Sugrs hve reltion to its fertiliztion potentil. Theriogenology, een used extensively s supplements to extenrs to 4: improve sperm motility during ryopreservtion of 4. Suquet, M., C. Drenno, C. Fuvel, J. Cosson nd sperm in severl fish speies suh s Afrin tfish R. Billrd, 00. Cryopreservtion of sperm in mrine (Clris griepinus) [22, 23] nd vrious sturgeon fish. Aquulture, 31: speies [12, 14]. Optiml extenr for freezing of fish. Snsone, G., A. Froini, S. Ieropoli, A.L. sperm vries mong fish speies. Sline solutions re Lngellotti, M. Ointe nd D. Mtssino, 02. wily used for ryopreservtion of Cyprini speies Effets of extenr omposition, ooling rte nd [24, 2]. However, sline solutions hve lso een freezing on the motility of Se ss (Dientrrhus reported to e less effiient extenrs in omprison lrx L.) spermtozo fter thwing. Cryoiology, to shris or rtifiil seminl plsm for 44: ryopreservtion of sperm in Afrin tfish 6. DeGrf, J.D. nd D.L. Berlinsky, 04. Cryogeni nd (C. griepinus) [23], Common rp (C. rpio) []. In this refrigerted storge of Rinow smelt (Osmerus experiment, postthwed sperms with MeOH mordx) spermtozo. Journl of Applied onentrtion of 10% hs the highest moility nd Ihthyology, 3: motility durtion nd this ws ontrst to the results 7. MrtínezPármo, S., S. PérezCerezles, F. Gómezotined y Shigueki Ysui et l. [26]. These reserhers Romno, G. Blno, J. A. Sánhez nd M.P. Herráez, hve Announed tht the mximum motility nd motility 09. Cryonking s tool for onservtion of durtion of postthwed sperm of Misgurnus iodiversity: Effet of Brown trout sperm nguilliudtus ws with MeOH onentrtion of 1%. ryopreservtion on the mle geneti potentil. [27] reported tht the most suitle ryoprotetnt for Theriogenology, 71: sperm ryopreservtion of Jv Br (Bronymus 8. Lhnsteiner, F., T. Weismnn nd R. Ptzner, 199. gonionotus), ws with MeOH 10%. A uniform method for ryopreservtion of semen of The reson of differene in suitle nsity of the slmonid fishes: Onorhynhus mykiss ryoprotetnt in written result with experiment result my (Wlum), Slmo trutt frio L., Slmo trutt e for seleted speies, differene in extenr solution or lustris L., Coregonus sp. Aquulture Reserh, semen is speifi hrteristis of this speies. 26: Cho, N.H. nd I.C. Lio, 01. Cryopreservtion of CONCLUSION finfish nd shellfish gmetes nd emryos. Aquulture, 49: 789. From the ove study it n e onlud tht 10. Ciereszko, A., G.P. Toth, S.A. Christ nd K. storge of frozen semen hs negtive impt on motility Drowski, Effet of ryopreservtion nd durtion nd motility perentge of postthwed sperms. theophylline on motility hrteristis of lke Results lso showed tht upmost moility nd the motility sturgeon (Aipenser fulvesens) spermtozo. durtion of postthwed sperms were oserved from Theriogenology, 4: sperm frozen t extenr GFSE 2 with the onentrtion of 11. Billrd, R., J. Cosson nd O. Linhrt, 00. Chnges in MeOH 10%. the flgellum morphology of intt nd frozen/thwed Sierin sturgeon (Aipenser erii, REFERENCES Brndt) sperm during motility. Aquulture Reserh, 31: Vesogh, G.H nd B. Mostgeer, 199. Freshwter 12. Tsvetkov, K.I., J. Cosson, O. Linhrt nd R. Billrd, fishes. Press Tehrn University of Irn, pp: Motility nd fertilizing pity of fresh nd 2. Billrd, R., J. Cosson, G. Perhe nd O. Linhrt, 199. frozenthwed spermtozo in sturgeon, Aipenser Biology of sperm nd rtifiil reprodution in rp. erii nd Aipenser ruthenus. Journl of Applied Aquulture, 129: Ihthyology, 12:

5 World J. Fish & Mrine Si., (): , Irwn, H., V. Vthiphndhi nd S. Nimrt, 10.. Horváth, A., E. Miskolzi nd B. Urányi, 03. The effet of extenrs, ryoprotetnts nd Cryopreservtion of Common rp sperm. Aqut. ryopreservtion methods on Common rp Living Resour, 16: (Cyprinus rpio) sperm. Animl Reprodution 21. Hrvey, B., R.N. Kelley nd M.J. AshwoodSmith, Siene, 122: Cryopreservtion of Zer fish spermtozo 14. Glogowski, J., R. Kolmn, M. Szepkowski, using Methnol. Cn. J. Zool., 60: A. Horvth, B. Urnyi, P. Siezynski, 22. Steyn, G.J. nd J.H.J. Vn Vuren, The A. Rzemienieki, J. Domgl, W. Deminowiz, fertilizing pity of ryopreserved Shrp tooth A. Kowlski nd A. Ciereszko, 02. Fertiliztion rte tfish (Clris griepinus) sperm. Aquulture, of Sierin sturgeon (Aipenser erii) milt 63: ryopreserved with methnol. Aquulture, 23. Urnyi, B., A. Horvth, Z. Vrg, I. Mgyry nd 211: F. Rdis, Effet of extenrs on sperm 1. Routry, P., D.K. Verm, S.K.Srkr nd N. Srngi, ryopreservtion of Afrin tfish. (Clris 07. Reent dvnes in rp seed prodution griepinus, Burhell, 1822). Aquulture Reserh, nd milt ryopreservtion. Fish Physiol. Biohem, 30: : Linhrt, O., R. Billrd nd J.P. Proteu, Gwo, J.C., 00. Cryopreservtion of sperm of some Cryopreservtion of Europen tfish (Silurus glnis mrine fishes. In: Tiersh, T.R., Mzik, P.M. (Eds.), L.) spermtozo. Aquulture, 11: Cryopreservtion in Aquti Speies. World 2. Kwntong, S. nd A.N. Brt, 06. Short Aquulture Soiety, Bton Rouge, LA, pp: ommunition: Cryopreservtion of Blk er 17. Alvrez, B., R. Fuentes, R. Pimentel, Z. Ad, tfish, (Pngsius lrnudii, Boourt) sperm. E. Crer, E. Pimentel nd A. Arenl, 03. High fry Aquulture Reserh, 37: 997. prodution rtes using postthw Silver rp 26. Shigueki Ysui, G., L. ArisRodriguez, T. Fujimoto (Hypophthlmihtys molitrix) spermtozo unr nd K. Ari, 02. A sperm ryopreservtion protool frming onditions. Aquulture, 2: 191. for the Loh (Misgurnus nguilliudtus) nd its 18. Bsvrj, N. nd S.N. Heg, 04. ppliility for other relted speies. Cryopreservtion of the endngered mhseer Theriogenology, 4: (Tor khudree) spermtozo: I. Effet of extenr 27. Ainwnto, H., S. Rhyu nd R. Lestri, 13. omposition, ryoprotetnts, dilution rtio nd Cryopreservtion of Jv Br (Bronymus storge period on postthw viility. Cryoiology, gonionotus) Using Egg Yolk s Cryoprotetnt. 49: Glol Veterinri, 10: Rodin, M., D. Gel, M. Koour, S.M.H. Hdi, M. Hulk nd O. Linhrt, 07. Cryopreservtion of tenh, Tin tin, sperm: sperm motility nd hthing suess of emryos. Theriogenology, 67: