Keywords fish, genotype, rainbow trout, sperm.

Transcription

1 World Ademy of Siene, Engineering nd Tehnology Interntionl Journl of Bioengineering nd Life Sienes Vol:, No:, Quntittive Chrteristis of Rinow Trout, Onorhynhus Mykiss, Neo-Mles (XX Genotype) nd Super-Mles ( Genotype) Sperm Rdosłw K. Kowlski, Bet Srosiek, Wiesłw Deminowiz, Jędrek Judek, Krzysztof Goryzko, Stefn Doosz, Henryk Kuźmiński, Krystyn Demsk-Zkęś, Igor Bik, Jn Glogowski Interntionl Siene Index, Bioengineering nd Life Sienes Vol:, No:, wset.org/pulition/887 Astrt Rinow trout homogmeti mles, (XX or sex genotype), n e otined, respetively, through msulinistion of geneti femles or indued ndrogenesis. Aim of this study ws to ompre reprodutive potentil of neo-mles (XX) nd super-mles () with heterogmeti mles (). We mesured spermtozo motility prmeters, sperm onentrtion, osmollity nd hrterized protein profiles in smples of stripped nd testiulr sperm otined from nd mles, nd testiulr sperm of XX mles. The motile spermtozo, s mesured y oth sujetive method nd CASA, showed no differenes etween testiulr sperm of XX mles nd stripped sperm of nd mles wheres testiulr sperm of nd mles hd signifintly lower sperm motility. Result of protein densitometry showed similrities in protein profile etween seminl plsm of nd mles nd testiulr fluids of XX mles. Testis of XX mles showed speifi histologil strutures of ysts onsists hypertrophied Sertoli ells. Keywords fish, genotype, rinow trout, sperm. I. INTRODUCTION lmonid fish hve sex-determining system in whih Sgenotypes t single gene or region on lrgely undifferentited sex hromosomes determine phenotypi sex []. It is well known tht exogenous verterte steroid hormones pplied to immture fish hve potentil to reverse the phenotypi sex. Androgens will hnge sex in femle diretion wheres estrogens will rete opposite effet. Indued sex-reversl, nmely, msulinistion of XX sex genotypes, is mens to produe monosex ll-femle Rdosłw K. Kowlski, Bet Srosiek, Wieslw Deminowiz, Jędrek Judek, Jn Glogowski re with the Moleulr Andrology Group, Deprtment of Gmete nd Emryo Biology, Institute of Animl Reprodution nd Food Reserh, Polish Ademy of Sienes in Olsztyn, -77 Olsztyn, Tuwim, Polnd (phone: ; fx: ; e-mils: r.kowlski@pn.olsztyn.pl;.srosiek@pn.olsztyn.pl; w.deminowiz@pn.olsztyn.pl; j.judek@pn.olsztyn.pl; j.glogowski@pn.olsztyn.pl). Krzysztof Goryzko, Stefn Doosz, Henryk Kuźmiński re with Deprtment of Slmonid Reserh, Inlnd Fisheries Institute, Rutki, Zukowo, Polnd (emils: goryzko@infish.om.pl; doosz@infish.om.pl hkuzminski@infish.om.pl) Krystyn Demsk-Zkęś, Jn Glogowski re with Deprtment of Ihtiology, Wrmi nd Mzury University in Olsztyn, -97 Olsztyn, Polnd (emils: krysidz@uwm.edu.pl; j.glogowski@pn.olsztyn.pl) Igor Bik is with Reprodutive Biology Group, Fulty of Biosienes nd Aquulture, University of Nordlnd, 89 Bodø, Norwy (emil: igor.ik@hio.no) popultions of fish under sex-determining system, inluding slmonids. Typilly, the proess of msuliniztion is indued y orl dministrtion of ndrogens through feed in the erly stges of sex differentition []. Sex reversed femles (neo-mles) produe sperm upon sexul mturtion, whih n e used to produe tretment-free genertion of ll-femle XX individuls. All-femle trout show fster growth rte euse mturtion in femles strts lter thn in mles; therefore, the prodution effiieny n e improved when monosex system of prodution is employed []. In the rinow trout, the sperm duts of mny neo-mles do not form properly nd the lk or the vrious stge of sperm dut redution re n usul fter-tretment mlformtions [7]. It might e used y diffiulties in orl dministrtion of hormones t urte dose for thousnd of fishes t the sme time. This imply the wy of neo-mles exploittion, whih re sed on srifiing the fish nd susequent testiulr milt olletion diretly from the mlformed testis. Another mnipultion possile in fish is n ndrogenesis. The key of this proess is to indue the development of diploid emryo on the sis of mle geneti mteril. This tehnique hve gret potentil in se of nking sperm from endngered fish. In order to intivte the genome of ooytes X-rys or gmm rys re used [3]. Interruption of the lst stge of the first mitoti division leds to the integrtion of the two opies of hromosomes nd to form diploid nulei, whih develops homozygous individul. Indution of ploidity in rinow trout is usully hieved vi pressure shok [], [3]. In view of the growing quulture nd the widespred use of "rtifiil" insemintion in fish, it is neessry to monitor the qulity of sperm. The si prmeters of sperm qulity of fish re the sperm onentrtion nd motility s well s their fertiliztion ility. Rinow trout fertiliztion suess ws positively orrelted with sperm motility prmeters [], [8]. However in other slmonids it hs not een onfirmed [], [8], [3]. The reserh relted to sperm qulity of homogmeti rinow trout mles re sre nd should e enlrged s these form of fish eome populr in quulture prtie. The ojetive of this study ws to ompre the sperm of ndrogeneti mles (mles ) nd sex reversed femles (mles XX) with seperm from the ontrol group (mles ). Informtion out the qulity of gmetes otined from 3 sholr.wset.org/37-89/887

2 World Ademy of Siene, Engineering nd Tehnology Interntionl Journl of Bioengineering nd Life Sienes Vol:, No:, Interntionl Siene Index, Bioengineering nd Life Sienes Vol:, No:, wset.org/pulition/887 homogmeti fish might help in the plnning of prodution sed on fish otined y genomi or hormonl mnipultion. An importnt ojetive of this study ws n nlysis of the low moleulr weight protein omposition of seminl plsm to investigte the role of sperm dut in the omposition of seminl plsm protein s well s the influene of sex genotype on protein ontent of seminl plsm. II. MATERIALS AND METHODS A. Fish Fish for the study were produed nd held t the Deprtment of Slmonid Reserh, Inlnd Fisheries Institute, Rutki, Polnd under stndrd proedures. Control mles originted from Rutki strin. Androgeneti super-mles () originted from popultion otined s desried y [], [3]. In rief, eggs were irrdited with gmm rys (3 rds) in order to destroy mternl genome, nd susequently fertilized. At 3 min post fertiliztion t C, eggs were sujeted to hydrostti pressure shok (7 psi) for minutes in order to otin diploid emryo [8]. The resulting ndrogeneti progeny ws omposed of either femles XX or super-mles. The fish were rered until sexul mturity. Three yers old individuls were used for the study. Neo-mles (XX) were otined vi msuliniztion using 7-α-methyltestosterone t dose of 3 mg kg - of feed dministered for dys from the eginning of strt feeding []. Upon sexul mturtion, neo-mles were identified y the sene of sperm duts, often ompnied y presene of rudimentry femle gonds []. Fish for the study were t the ge. In totl, for the omprtive nlysis of sperm qulity prmeters, sperm smples were olleted from mles nd super-mles (stripped sperm). Additionlly, testis were disseted from 8 mles, 8 super-mles nd 3 neo-mles XX, to ollet testiulr sperm smples (Fig. ). Fig. Shemti of otining mteril for reserh B. Histology of gonds Smples for histology were tken in the middle of the reeding seson from mture mles. In totl, smples of testis were olleted from eh group of sex genotypes, XX, nd. Exised setions of testis were preserved in Bouin's fluid for 3 dys followed y rinsing three times with 7% ethnol, dehydrtion in ethnol grdients 7, 8, 9, 9, nd %, nd wshing three times in xylene. Smples were trnsferred to liquid prffin, then emedded in prffin loks. Bloks were ut with rotry mirotome Lei RM-type, using the trnsverse or longitudinl setions. Setions of thikness of µm were pled on slides. Prepred smples were sujeted to stining with hemtoxylin nd eosin (HE) or y Mllory. After drying, histologil preprtions were viewed using light mirosopy. C. Quntittive prmeters of sperm qulity Spermtozo motility ws evluted y sujetive method nd using CASA system. For sujetive method spermtozo motility prmeters were determined under light mirosope under the mgnifition x. For utomti sperm motility nlysis CASA system mnuftured y Hoson's Vision (Hoson Vision Ltd, Deryshire, UK) ws used. Sperm smples were diluted : (stripped sperm) or : (testiulr sperm) with n tivting medium uffer DIA3 [] efore nlysis. Immeditely fter dilution (s) the liquot of.7 µl ws pled on the wells printed mirosope slide with (eh well hd µm depth) nd sperm movement ws reorded under the lens with mgnifition of x within seonds. Then the tpe reordings were nlyzed using the Hoson Sperm Trker. Progrm settings for the imge nlysis t x ojetive mgnifition were: rdius=8. mm; predit = off; video = pl; spet =.9; refresh time = s; threshold /-; filter weightings =, =3; 3=3; =3, imge pture rte = Hz. We nlyzed the following prmeters: VSL - strightliner veloity of sperm movement (µm s - ), VCL - urviliner veloity of sperm movement (µm s - ), VAP totl veloity of sperm movement (µm s - ), STR - stritlinerity of sperm movement (%), BCF et ross frequeny (Hz), ALH - mplitude of lterl hed displement (µm), LIN linerity of spermtozo motility (%), MOT - the perentge of motile sperm (%). Conentrtion of spermtozo ws lulted y spetrophotometri method [9]. Smples of stripped sperm were diluted times with solution of.7% NCl. Smples of testiulr sperm were diluted times. Asorne ws mesured t wvelength of 3 nm. The results were red from lirtion urve prepred for rinow trout sperm [9]. Aout ml of sperm smples ws entrifuged ( min, 8 x g) to otin seminl plsm for iohemil nlysis. In the se of testiulr sperm smples, the testiulr fluid remining fter entrifugtion of sperm ws used for nlysis. Totl protein ontent in the seminl plsm nd testiulr fluid were determined ording to Lowry et l. (9), using ovine serum lumin (BSA) s stndrd protein. Seminl 3 sholr.wset.org/37-89/887

3 World Ademy of Siene, Engineering nd Tehnology Interntionl Journl of Bioengineering nd Life Sienes Vol:, No:, Interntionl Siene Index, Bioengineering nd Life Sienes Vol:, No:, wset.org/pulition/887 plsm nd testiulr fluids osmollity were mesured using the WESCOR Vpor Pressure osmometer nd the results were expressed in mosm kg -. Tris-triine SDS-PAGE ws performed in % of T,3% C peptide polyrylmide gel nd % T, 3% C trnsition polyrylmide gel [39]. For the omprison of protein nds etween seminl plsm nd testiulr fluids (n= in eh group) equl mount of protein ws pplied for eletrophoresis nd mixed with uffer ontined. M Tris-HCl, % glyerol, % SDS,.3 M DTT with Coomsie Brillint Blue, ph.8. Smples were oiled ( C) for min.. Eletrophoresis ws performed using n node uffer (): mm Tris-HCl, ph 8.9 uffer nd thode (-): mm Tris-HCl, mm Triine,.% SDS. The voltges pplied were 3 V - t thikening gel, V - t gel trnsition, nd 8 V until the proteins rehed the end of the gel. After seprtion, the gel ws pled for 3 minutes in fixtive (% methnol % eti id) nd trnsferred to dye (.% Coomsie Brillint Blue). Gels were deolorized in % eti id within h. Phosphte isomerse (MW. kd), myogloin (MW.9 kd), α-ltolumin (MW.37 kd), protinin (MW. kd), insulin (MW 3.9 kd), nd ktrin (MW.3 kd; ll BioRd, USA) were used s moleulr weight stndrds. Anlysis of eletrophoregrm densitometry ws performed to estlish the proportion of the seminl plsm/testiulr fluid proteins in eh of tested sex genotypes. The study inluded eletrophoreogrms stined for protein otined during the eletrophoreti seprtion. We determined reltive optil density (RGO) of ll protein nds visile on the gels using softwre Kodk D (Moleulr Imgin System, Estmn Kodk Compny, USA). D. Sttistil nlysis Dt were nlysed using GrphPd Prism softwre (Pizzoni J. Estmn Kodk, New Hven, CT, USA). Dt representing perentge of motile spermtozo were trnsformed prior sttistil nlysis with use of rsine trnsformtion. ANOVA followed y Tukey's post-ho omprison test ws used to evlute sttistilly importnt differene etween nlyzed groups of mles. T-test for pired smples ws used to ompre protein ontent nd the protein profiles etween seminl plsm nd testiulr fluids. III. RESULTS Sexully mture testis of ontrol mles were uilt of mny loules (from to µm in dimeter) filled with sperm. The wlls of loules were uilt of onnetive tissue - µm thik. The outer wll of the testis ws - 3 µm thik (Fig.,). Sperm duts hd multiple memrnous strutures involved towrds the enter (Fig. ). The interior of the sperm dut ws filled with sperm nd its wlls ws overed y epithelil ells (Fig. d). Fig.. Histology of testis (, ) nd sperm dut (, d) of rinow trout dult mle t the pek of reprodutive seson. S - spermtozo, CT - onnetive tissue, W - the outer wll of the testis, E epithelium In the testis of super-mles loules were visile ( - 3 µm in dimeter) filled with sperm. The onnetive tissue forming the wlls of entriloulr ells ws visile. The outer wll of the testis hd thikness of - µm (Fig. 3,). Spermtogonil ells were loted t distl res of loules (Fig. 3). The enter of sperm dut ws filled with sperm nd its wlls lined with epithelil ells (Fig 3, d). Fig. 3 Histology of testis (, ) nd sperm dut (, d) of rinow trout dult super-mle t the pek of reprodutive seson. S - spermtozo, SG - spermtogoni, CT - onnetive tissue, W - the outer wll of the testis, L lumen of the sperm dut, E epithelium Testis of neo-mles XX were uilt of lrge loules (out mm in dimeter) loted in the entre nd smller loules (out µm) loted in the outer prts of the gond (Fig., d, e, f). The thikness of the outer ell wll rnged from 3 to µm.. Cysts of mturing sperm were visile in the onnetive tissue of the loules (Fig., d). Centriloulr ell wlls nd spermtogoni surrounded y Sertoli ells were oserved in the outer prts of the testis (Fig. e, f). In the 37 sholr.wset.org/37-89/887

4 World Ademy of Siene, Engineering nd Tehnology Interntionl Journl of Bioengineering nd Life Sienes Vol:, No:, inner prts of testis, ysts were visile omposed of hypertrophied Sertoli ells (Fig e, f). the highest vlues in smples of stripped sperm mles (3.%). In testiulr sperm of XX mles this vlue ws lose to the vlue mesured for stripped nd testiulr sperm of mle (respetively.9,.8 nd 9.%). VCL (μm/s) 8,7 7,, 9, 7, VSL (μm/s) ,7 3,3,7 9, 3, Interntionl Siene Index, Bioengineering nd Life Sienes Vol:, No:, wset.org/pulition/887 Fig. Histologil strutures of rinow trout dult neo-mle XX t the pek of reprodutive seson;, - outer prt of the testis;, d, e, nd f - the internl prt of the testis. S - sperm, SG - spermtogoni, ST - Sertoli ells, CT - onnetive tissue, C - yst of mturing sperm, W - the outer wll of the testis, CH - yst of the hypertrophied Sertoli ells, HS - hypertrophied Sertoli ells. Sperm motility (MOT) ws the highest in smples of the stripped sperm of nd mles nd the testiulr sperm of XX mles nd it ws out 7%. Testiulr sperm nd mles ws hrterized y low sperm motility (respetively 8. nd 33.%). Sperm urviliner veloity (VCL) in sperm smples stripped from mles (verge.7µm s - ) ws signifintly higher thn in other tested groups (Fig. ). VCL of testiulr sperm otined from XX neo-mles ws on verge 7.µm s - nd ws similr to the vlues mesured for stripped sperm of mles. The highest strit liner veloity (VSL) nd totl Speed (VAP ws mesured for the stripped sperm from the nd. In oth groups testiulr sperm showed sttistilly signifint lower vlue of VSL nd VAP (P<). The vlue of STR ws signifintly higher in testiulr sperm ompred to stripped one, oth in the mle nd (P<). The BCF did not differ signifintly etween the groups nd rnged from.78 Hz (testiulr sperm of XX mles) to.8 Hz (stripped sperm of mles). ALH ws the highest in the stripped sperm of mles nd it ws.9µm. The lowest vlue of ALH ws found in stripped nd testiulr sperm of mles (respetively. nd 9.µm). For oth group of mles nd, ALH vlues mesured in stripped sperm were signifintly higher thn the vlues mesured in testiulr sperm (P<). The linerity of sperm motility (LIN) rehed VAP (μm/s) BCF (Hz) LIN (%) ,3,8,8 7,3, 9, 79,, 3,,7,79 3, 7,,78,9 STR (%) ALH (μm) 3 8 8,9, ,7 Fig. Motility prmeters of stripped nd testiulr sperm otined from rinow trout mles with sex genotypes,, nd XX. Different letters indite sttistilly importnt differene etween men vlues (P<.) Similrly to CASA results, sperm motility mesured y sujetive method ws the highest in stripped sperm of mles nd super-mles, nd lowest in testiulr sperm smples of nd mles. Intermedite vlues of MOT, not differing signifintly from other groups, were found for testiulr sperm smple otined from XX neo-mles (Tle I). The lowest spermtozo onentrtion ws found in stripped sperm from super-mles (3.89 x 9 ml - ), signifintly lower thn the vlue of spermtozo onentrtion of stripped sperm from mles (.37 x 9 ml - ). The onentrtion of spermtozo in ll testiulr sperm smples ws signifintly higher nd it ws over x 9 ml -. (Tle I). Osmollity of seminl plsm nd testiulr fluid rnged MOT (%) 8,8,9 8,,, 77,9 7,3 d 9, 33,,, 73, 38 sholr.wset.org/37-89/887

5 World Ademy of Siene, Engineering nd Tehnology Interntionl Journl of Bioengineering nd Life Sienes Vol:, No:, Interntionl Siene Index, Bioengineering nd Life Sienes Vol:, No:, wset.org/pulition/887 from to 38 mosm kg -. The lowest osmollity ws found in smples of super-mle seminl plsm. The totl protein ontent ws lowest in seminl plsm of mles (.7g l - ). The highest protein ontent of sperm ws found in testiulr plsms otined from oth, nd, mles (respetively 3.3 nd.9 g l - ; Tle I). TABLE I BASIC PARAMETERS OF STRIPPED AND TESTICULAR SPERM QUALITY OBTAINED FROM RAINBOW TROUT MALES HAVING SEX GENOTYPES,, AND XX neomles Mles Super-mles XX Sperm Sperm Testis Testis Testis dut dut n=8 n=8 n=3 n= n= Motility 3 9 (%) ±9 ± ±8 ± ± Sperm onentrtion ( 9 ml - ±.9 ±. ±.9 ±. ±. ) Osmollity (mosm kg - ) ± ± ±3 ± ± Protein d onentrtion (g l - ±.3 ±.3 ±.7 ±3.77 ±. ) Averge vlues mrked with different letters differ signifintly (P <.) from eh other in line. Reltive optil density Reltive optil density kd kd testis sp sp Reltive optil density Reltive optil density kd Fig. SDS PAGE eletrophoresis of seminl plsm/testiulr fluid proteins in denturing grdient triine gel. Densitogrms of seminl plsm (sp) nd testiulr fluid (testis). The X-xis shows the moleulr weight. In the seminl plsm of mle nd were reltively more protein with moleulr weight 39.3, 3.7, 3. nd.3 kd ompre to testiulr fluids (Tle II). Proteins of 3. kd moleulr weight were the most undnt in protein profiles in ll genotype groups tested. Their ontent ws reltively the highest in the seminl plsm of stripped sperm from nd mles, s well s in testiulr sperm of XX mles. Testiulr fluids of nd mles showed higher reltive optil density of proteins., 7.,., 3.9, nd 7.3 kd. 7 8 kd testis sp TABLE II RELATIVE OPTICAL DENSITY (RGO) OF PROTEIN BANDS IN RAINBOW TROUT SPERM IN DENATURING GRADIENT TRICINE GEL; SEMINAL PLASMA (SP) AND TESTICULAR FLUID (T) WERE OBTAINED FROM MALES OF, XX, AND SEX GENOTYPES No Bnds (D) sp n=..9 ±..3 ±..39 ±. sp n= t n= XX t n= Reltive optil density (±SD).. ±.9.. ± ±.. ±..33 ±..7 t n= IV. DISCUSSION For the first time, we hve mde omprtive study of sperm qulity in ll possile sex genotypes in rinow trout. Results of sperm motility CASA prmeters, sperm onentrtion, protein profile, nd histology were olleted nd ompred etween the three genotypi group of rinow trout mles (, nd XX). We onfirmed tht testis of XX mles posses some ellulr strutures omposed from hypertrophied Sertoli ells whih ws not present in nd. Our results indite tht testiulr sperm otined from XX mles hve similr qulity to those otined from ±...3. ±..7.3 Averge vlues mrked with different letters differ signifintly (P <.) from eh other in line. Bnds hving ROG not exeeding re mrked s ; sene of nd is mrked s sholr.wset.org/37-89/887

6 World Ademy of Siene, Engineering nd Tehnology Interntionl Journl of Bioengineering nd Life Sienes Vol:, No:, Interntionl Siene Index, Bioengineering nd Life Sienes Vol:, No:, wset.org/pulition/887 nd mles fter stripping. Also protein profile of XX testiulr sperm ws similr to those oserved in seminl plsm of nd mles. Putting ll together we ssume tht sperm otined from neo-mles testis hve similr hrteristis to stripped sperm of nd mles nd higher thn testiulr sperm of nd mles. Setions of sperm duts of mle nd super-mle reveled the existene of speifi folded struture of the epithelium. Sperm dut in teleost fish hs seretory funtion [3], [3]. It regultes, mong others, ion onentrtion nd ph of seminl plsm []. It ws shown in slmonids tht the sperm dut plys importnt role in quisition of sperm motility potentil [3]. The folded struture n inrese seretory re of the sperm dut of mles nd. This might e relted to visile firous seretion in sperm dut lumen s ws desried elsewhere [3]. Histologil strutures of testis of nd mles were similr to testis of Atlnti slmon (Slmo slr) nd rown trout (Slmo trutt m. trutt) [7]. Testis of XX neo-mles differed from the testiulr histology imge of nd mles. The ysts of epithelil hyperplsi were found. This tissue, filling loule, proly onsists of hypertrophied Sertoli ells. Similr ellulr strutures were found in the testis of gldiolus (Xiphophorusmultus), sujeted to hormonl mnipultion []. Also in neo-mles of rinow trout suh struture ws lredy reported []. Imge of firous seretions on the surfe of these ysts might indite, the exretory funtion of these strutures. Sperm of most teleost fish is hrterized y strightliner motility, whih is refleted y nerly identil vlues of VSL nd VCL []. In rinow trout, VSL vlues re usullly muh lower thn VCL vlues, whih results in low LIN (LIN = VSL/VCLx%) [3], []. In the present study, the highest vlues of LIN were found in stripped sperm of super-mle. The vlues of ALH mesured in stripped sperm of super-mles were similr to those found for stripped sperm of rinow trout (out 8. µm) []. However, in our work ALH vlue of smples otined from mles were lmost twie higher (Fig. f). A possile explntion for this divergene of ALH results is the influene of ge nd line of fish used for the experiments. Referenes [3] studied mles from utumn-spwning strin of (ge 3, wheres in the present study, sperm from fish from springspwning strin of ge nd ws exmined. The low vlue of the VSL nd LIN, omined with high rtes of ALH in the se of mmmlin sperm indited hypertivity []. In the se of freshwter fish sperm movement of suh nture n e mnifesttion of hypoosmoti shok ourred prior to fertiliztion [38]. The et ross frequeny (BCF) ws similr in ll groups of mles (Fig. ). The vlue of this prmeter, - Hz, ws similr to the vlues reported y other reserhers []. It seems tht the vlue of BCF is stle feture in rinow trout sperm movement hrteristis nd it does not hnge during the finl mturtion in spermti dut. VCL (urviliner veloity) is n importnt ftor in fish mle reprodutive suess [9], [8]. VCL in stripped sperm smples of mles ws on verge.7 µm s -, whih is higher thn vlues reported efore for rinow trout. Referenes [3], [], [8] reported VCL rnging from to µm s -. This differene might e results of using different genetilly fish stok or different mesurements tehnique. The lowest vlues of VCL were found in testiulr sperm of nd mles. Surprisingly, testiulr spermtozo of XX neo-mles hd signifintly higher vlue of VCL. This might indite tht while sperm of neo-mles re olleted from the testis, their struture nd physiology re different from tht in norml mles resulting with differene in sperm mturity. The highest perentge of motile sperm (MOT) ws found in the stripped sperm of mles, super-mles, nd in testiulr sperm of XX neo-mles (73.7, 77.9 nd 73.%, respetively). MOT in testiulr sperm of nd mles ws signifintly lower (8. nd 33.%, respetively) Our results re in greement with erlier oservtions inditing tht testiulr sperm of XX neo-mles showed motility rtes similr to smples stripped from mles []. Tking into ount the two most importnt prmeters of sperm, VCL nd MOT, it n e onluded tht the est qulity sperm ws stripped sperm from mles, super-mles nd testiulr sperm of XX mles, wheres testiulr sperm from mles nd super-mles hd signifintly poorer qulity. This oservtion imply tht physiology of neo-mles testis is quite different from tht in norml mles testis. Further study re neessrily to investigte if the XX mles gonds hve some prt of spermti dut potentil in term of induing mturtion of spermtozo. The testiulr sperm in ll groups of mles hd onentrtion higher thn x 9 ml -. A similr onentrtion of sperm in the testis of rinow trout nd XX mles were reported []. The onentrtion of sperm in smples otined from super-mles ws lmost hlf less thn the onentrtion of spermtozo in mles (on verge 3.89 x 9 ml - versus.37 x 9 ml - ). Sperm onentrtion vlues for rinow trout mles reported y other reserhers, vry from x 9 ml - [], [3] 3. x 9 ml - [7]. The differenes in sperm onentrtion my e n effet of the differenes etween lines of rinow trout spwning in utumn or spring []. Usully lower thn optiml for speies sperm onentrtion is onneted with poor qulity of mles gmetes []. However in se of mles their CASA prmeters ws similr to those from ontrol group exept ALH. It seems tht low sperm onentrtion in these fish might e n result of inreed used y speifiity of tehnique used for otining ndrogeneti mles. The osmoti pressure of seminl plsm my e n inditor of ontmintion y urine whih might redue the osmollity (s their ntive osmollity re lower thn 7 mosm) nd onsequently deline sperm motility nd its fertilizing ility [], [], [8], [37]. Our results showed tht seminl plsm osmollity hd the lowest vlue in smples 3 sholr.wset.org/37-89/887

7 World Ademy of Siene, Engineering nd Tehnology Interntionl Journl of Bioengineering nd Life Sienes Vol:, No:, Interntionl Siene Index, Bioengineering nd Life Sienes Vol:, No:, wset.org/pulition/887 otined from super-mles. However, low osmollity vlues were not ompnied y lower motility whih might suggest tht it ws not result of urine ontmintion. Low osmollity ws linked with lower protein ontent in seminl plsm of mles ompred to mles. The protein ontent in seminl plsm of rinow trout rnges from.7 to.8 g l - []. In our study, seminl plsm of mles ontined. g l - of totl protein on verge, similrly to the other dt []. In the se of super-mles, seminl plsm protein ontent ws muh lower (.7 g l - ). Low protein ontent in the se of mles ws ompnied y low onentrtion of spermtozo. The low sperm onentrtion nd protein ontent in ejulte might e speifi feture of mles. Testiulr sperm protein ontent ws 3.3 g l - nd.9 g l - for mles nd, respetively, wheres in the se of XX neo-mles it ws lower (7. g l - ). Testis of XX neomles showed the presene of lrger loules, ompred with nd mles (Fig. ). Therefore, they hve less onnetive tissue in the testis thn the other groups of mles. Sine the mertion of the testis my use dmge to loules wlls, protein present in the seminl plsm my e prtilly derived from dmged tissues. This my e more signifint for testiulr fluid otined from the smples of nd mles thn XX mles. Referenes [9] showed tht low moleulr weight protein of seminl plsm my positively ffet sperm motility. Differenes in the numer of low moleulr weight protein nds found in the present study re generlly ssoited with differenes etween seminl plsm nd testiulr fluids of nd mles. Sperm dut n e soure of some proteins due to ft tht some of them re more undnt in seminl plsm thn in testiulr fluid, s in the se low moleulr weight proteins [9]. A positive effet of seminl plsm protein frtion with moleulr weight elow kd on sperm viility were postulted [9]. Perhps they re prt of onditioning environment of the sperm mturtion. Their greter ontriution in the seminl plsm protein profile shows sperm dut s the dditionl soure (outside the testis) of protein synthesis. Slmonid spermtozo quire the ility to move during pssing through the sperm dut [3]. Testiulr sperm is therefore not finlly mtured. It is devoid of neessry omponents of seminl plsm produed in the sperm dut [33]. This might e refleted in the perentge of motile sperm from slmonid testis, lower thn in the stripped sperm [8], []. Also in our study, sperm motility of testiulr smples otined from mles nd super-mles ws low. However, sperm motility in testiulr smples of XX neomles did not differ signifintly from the motility of stripped sperm of nd mles. Also other uthors reported lower perentge of motile sperm from testis of rinow trout ompred to the motility of testiulr sperm of XX mles []. The differene in the hrteristis of testiulr sperm etween XX neo-mles nd mles hving developed sperm dut n e used y the ft tht t the dissetion of mture testis from or mles, lrge prt of finlly mture spermtozo is lredy relesed into the sperm dut. Therefore the remining sperm, lthough most of whih hs lredy ompleted the proess of spermtogenesis (s onfirmed y testiulr histology), hs not yet quired the full ility to move (lk of finl mturtion stge). The sperm motility rte of testiulr smples of XX neomles, s mesured y oth sujetive method nd CASA, ws similr to sperm motility mesured in stripped sperm of nd mles. It might e result of ompenstory (for sperm dut) struture found in XX mles testis proly derived from hypertrophied Sertoli ells []. It is possile tht ysts of hypertrophied Sertoli ells n regulte the omposition of testiulr plsm nd synthesize proteins, homologous to tht from sperm dut. 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