Effects of three different sources of pituitary extract on gonadal inducer in male and female pacu (Piaractus (

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1 Effects of three different sources of pituitry extrct on gondl inducer in mle nd femle pcu (Pirctus ( mesopotmicus) Dnilo Pedro Streit Júnior 1, Gentil Vnini de Mores 1 *, Ricrdo Pereir Ribeiro 1, Edurdo Shigueiro Skguti 1, Jyme Aprecido Povh 1 nd Heden Luiz Mrquez Moreir 2 1 Progrm de Pós-grdução em Zootecni, Universidde Estdul de Mringá (PPZ/UEM). 2 Deprtmento de Zoologi e Genétic, Universidde Federl de Pelots (UFPel). Author for correspondence. e-mil: gvmores@uem.br ABSTRACT. Mle nd femle pcus (Pirctus mesopotmicus) were induced with broiler chicken (BCPE), rbbit (RPE), nd crp (CPE) pituitry extrcts. The prmeters for spermtic vigor, progressive motility, fertiliztion nd htching rte, ccording to semen origin, did not show ny differences mong the tretments (P>0.05). RPE produced the lowest (P<0.05) semen volume nd the highest spermtozo number nd concentrtion (P<0.05). No difference ws observed (P>0.05) in the totl rte of bnorml spermtozo mong the tretments, but the secondry pthologies were higher (P<0.05) in the tretment with RPE. The femles induced with RPE did not present stimulus for htching. No differences (P>0.05) between BCPE nd CPE were found for ccumulted therml unit, number of oocytes/spwning grms, fertiliztion nd htchery rtes ccording to tretment origin. The result indictes tht BCPE use is recommended for gondl induction of P. mesopotmicus. Key words: quculture, fish, htching, semen, spermtozo, oocytes. RESUMO. Efeito de três diferentes fontes de extrto de pituitáris n indução gondl em mchos e fêmes de pcu (Pirctus mesopotmicus). Mchos e fêmes de (Pirctus mesopotmicus) form induzids com extrto de hipófise de frngo de corte (BCPE), coelho (RPE) e de crp (CPE). Os prâmetros vigor espermático, motilidde progressiv, tx de fertilizção e eclosão de cordo com origem do sêmen, não presentou diferenç (p>0,05) entre os trtmentos. O trtmento com BCPE produziu o menor volume de sêmen (p<0,05), o número e concentrção de espermtozóides form os mis ltos (p<0,05). Não foi observdo diferenç (p>0,05) pr o tx de espermtozóides com normliddes nos trtmentos, ms s ptologis secundáris form elevds (p<0,05) no trtmento com RPE. As fêmes induzids com RPE não presentrm estímulo pr desov. Não houve diferenç (p<0,05) entre BCPE e CPE pr unidde térmic cumuld, número de ovócitos liberdos/g de desov, tx de fertilizção e desov de cordo com origem dos trtmentos. O resultdo indic que o uso de BCPE é recomenddo pr indução gondl do P. mesopotmicus. Plvrs chve: qüicultur, peixe, desov, sêmen, espermtozóides, ovócitos. Introduction The serch for ntive fish with good niml science potentil hs resulted in pcu (Pirctus mesopotmicus) being one of the most utilized species for reserch in Brzil. Among its niml science chrcteristics, the high cpcity of survivl in cptivity my be quoted (Cstgnolli, 1992). The P. mesopotmicus is migrtory species in fresh wter nd, when in confined environment, it depends on hormonl induction to reproduce successfully (Cstgnolli, 1992). The first studies on pcu reproduction were crried out in the seventies, using humn Chorionic Gondotropin (hcg) s gondl inducer (Godinho et l., 1977). During the eighties, prtilly purified gondotropin nd slmon pituitry extrct were used by Cstgnolli nd Donldson (1981) in pcu reproduction (Pirctus mesopotmicus). Romgos et l. (1985) induced the Pirctus mesopotmicus with crude slmon pituitry extrct

2 440 Streit Júnior et l. nd hcg, nd Godinho nd Godinho (1986) used crp pituitry extrct. Mny dvntges justify the widespred use of pituitry method, when compred to other hormonl induction methods. According to Donldson nd Hunter (1983), the pituitry method is simple nd prcticl. Avin pituitry extrct hs lso been tested s possible fish gondl inducer (Amrl Jr., 1995; Yu et l., 1995; Brroso, 1999). Therefore, the purpose of this reserch ws to study the efficiency of broiler chicken nd crude rbbit pituitry extrct in the reproduction of mle nd femle pcu (P. mesopotmicus), compred to crp crude pituitry extrct, in order to find new, low-cost lterntive to trditionl crp pituitry extrct. Mteril nd methods Two experiments were crried out on the reproduction of pcu (P. mesopotmicus), from December 2001 to Jnury 2002, in the Fishery Sttion of Universidde Estdul de Mringá/Codpr, district of Florino, Mringá, Stte of Prná, southern Brzil. Seventy-eight P. mesopotmicus were selected (42 mles nd 36 femles) nd stocked into 600 m²tnk, with 0.5 Kg/m² density. During six months, these nimls received extruded rtion (d libtum), extruded feed with 38% protein, 7% crude fiber, 4% ether extrct, 13% dry mtter, 4.5% clcium, 0.8% phosphorus, nd 12% wet. Mles nd femles with pprent potentil for induction were selected. The mles relesed semen when their bdomen ws pressed. The femles presented convex belly, soft bdomen nd red urogenitl orifice. Fish were mrked in the lbortory on the bck fin with telephone wire nd divided into groups, ccording to sex. Ech tnk hd fifty-centimeterwter column in constnt wter flow, wter temperture t 25.5±1.5ºC, nd 4 to 7 mg/l of dissolved oxygen kept by the wter flow or through compressed ir from n utomtic compressor. Pituitry collection Broiler chicken pituitries were obtined from 45-dys nimls (men ge) from slughterhouse in Mringá. Rbbit pituitries were collected from nimls slughtered t the Experimentl Frm of the Universidde Estdul de Mringá (UEM), in Igutemi, Stte of Prná. The rbbits were 60 to 70 dys old, suitble ge for slughtering this species. Crp pituitry ws cquired in the mrket, redy to be used. Broiler chicken nd rbbit pituitries were obtined from n initil incision in the occipitl region, crossing between the eye nd the er, reching the center of the mouth. The niml brin nd the sell turcic were exposed nd the pituitry ws extrcted. In the birds, the incision ws mde with shrp knife; in the rbbits, with butcher s sw. Odontologicl curettes or injection needles (25X8) were used to extrct pituitry from the sell turcic. After extrction, the rbbit nd broiler chicken pituitries were plced into glss recipients filled with pure cetone, nd remined totlly immersed for ten hours. The cetone ws then chnged, nd the mteril ws kept for nother ten hours. After tht, the pituitries were weighed, plced on filtering tissue, nd tken into desicctor. The crp pituitry extrct used in the experiment ws obtined from trusted supplier of UEM fish frming sttion. Administrtion of pituitry extrcts Fish were selected nd trnsported to the lbortory, where they received the respective pituitry extrcts. The pituitries were mcerted in 100 ml mortrs nd physiologicl solution of 0.7% of NCl, in proportion of 4-mg pituitry per 1 ml-sline solution (Woynrovich nd Horváth, 1983). The crp pituitry extrct dosge utilized ws recommended by Woynrovich nd Horváth (1983) for species tht hve completed spwning. For chicken pituitry extrct, fllow-up dosge ws recommended by Brroso (1999). When rbbit pituitry extrct ws used, the dosge ws bsed on pilot work nd lte experiments with crp. The procedure of induction ws repeted four times for femles nd five times for mles, using the schedule indicted in Tble 1. Tble 1. Schedule of ppliction of bird, rbbit nd crp pituitry extrcts (mg/kg of living weight) in P. mesopotmicus. PITUITARY EXTRACT SEX DOSE CARP BROILER RABBIT CHICKEN MALE (42) 1 SINGLE FEMALE (36) 1 1 st nd Number of nimls used into prenthesis. One by one, the mles were tken out of the mnipultion tnks nd plced on 30-density fom mttress nd heds covered with wet cotton towel. A 5 ml syringe, with 27x7 needle, ws used to proceed the hormone injection, vi intr-peritonel. The femles received two pituitry extrct pplictions with n intervl of 12 hours between

3 Pituitry extrct on Pirctus mesopotmicus 441 ech one. Wter temperture ws constntly observed with thermometer of Celsius grdution. After reching 260 of ccumulted therml unit (ATU) (Ceccrelli et l., 2000), the femle pcu received light bdominl pressure. The ATU ws determined every hour, fter the lst hormonl ppliction until spwning time (Ceccrelli et l., 2000). When 260 ATU ws reched, the mles received bdominl pressure to relese the sperm. The mle fish received single dose of pituitry extrct, four hours before the lst hormonl dministrtion in the femle, in order to do the spermogrm. Experiment I Observtion of quli-quntittive quntittive prmeters of mle nd femle gmetes Ech niml ws tken out of the tnks to hve their gmetes collected. Following tht, the genitl region nd the nl fin of the fishes were dried with tissue pper nd the extrusion mde by light bdominl pressure. Oocytes were collected into plstic bowl nd semen into glss, properly identified nd trnsferred into syringes of severl sizes, in order to mesure their volume nd proceed with the next nlysis. After tht, the mles nd femles were tken bck to their tnks. From the collected semen of ech pcu, the volume, sperm concentrtion, progressive motility, spermtic vigor, morphopthology, nd totlity of spermtozo collection were nlyzed (Sorensen Jr., 1979). The summries of the procedure re shown below: Volume: bdominl mssge ws used until semen libertion occurred, which ws mesurble in syringes of severl sizes. Concentrtion of spermtozo: the semen ws diluted in smll beker, using the Shlli s pipette (0.02 ml) in 40 ml of buffered forml sline (Hncock, 1951), resulting in the dilution of 1:2000. After dilution, the Neubuer counting ws crried out in phse contrst microscopic, t 40X. Progressive motility nd spermtic vigor: semen drop ws diluted with eight distilled wter drops t room temperture in slide of optics microscopy. Afterwrds, it ws tken to the microscope of phse contrst, mgnified 40X nd nlyzed through subjective method, both vrints. For the progressive motility vrint, score from 0 to 100% ws used nd, for the spermtic vigor, score from 0 to 5 points. Morphology: for this nlysis, two smers were prepred on microscopic slides (Brth nd Oko, 1989) by diluting the semen in buffered forml sline in proportion of 1:2000 (semen/diluted solution, respectively). The smers were stined by Bengl Rose method (Conn, 1918), dried nd tken to the microscope of phse contrst mgnified 40X spermtozo were counted mong ech slide with smers of liberted semen of ech niml. Bent til, coiled til, crooked til nd smll hed were considered primry pthology. Shoehook, hedless, tilless nd proximl cytoplsmtic drop were considered secondry pthology. With the vlues of individul seminl volume, sperm concentrtion nd the totl spermtozo number/semen relese ws clculted. After the extrusion of ech femle, the oocytes were weighed in scles of 400 g, with sensibility of two shells, in order to withdrw three one-grm smples nd verify oocytes productivity (quntity of oocytes/g of spwning). The experimentl design ws completely rndom, with three tretments rrnged in fctoril of three hormones nd four repetitions (weeks) for femle nd five repetitions (weeks) for mle per tretment. Ech niml ws considered n experimentl unit. The semen volume, sperm concentrtion, totl number of sperm, progressive motility, spermtic vigor, spermtic morphopthology, spwning rte, verge number of spwning oocytes/g, nd ATU were nlyzed seprtely, ccording to the sttistic model below: Yijk = µ + Hi + Sj + HSij + εijk Yijk = observtion in reltion to mle or femle (k), receiving the hormone (i) in the week (j); µ = generl constnt; Hi = hormone effect; Sj = week effect; HSij = hormone interction (i) nd the week (j); εijk = rndom mistke ssocited to the observtion of niml (k) received hormone (i) in the week (j); Experiment II Evlution of reproductive performnce Fertiliztion nd htching rtes, referring to pituitry extrct used in both sexes were evluted in experiment II. After extrusion of oocytes or semen of the niml treted ccording to pituitry extrct doses, s indicted in Tble 1, fertiliztions were performed. The spwn ws collected in clen, dry nd identified plstic bowls. After tht, the semen ws put over the spwning nd goose fether crefully homogenized the ov. A new fether ws used for ech spwning. Every spwning (femles induced with crp, chicken

4 442 Streit Júnior et l. nd rbbit pituitry extrct) ws divided in three equl oocyte portions nd fertilized with semen of mles induced with crp, chicken nd rbbit pituitry extrct. This resulted in nine tretments (oocyte combintion X semen). The ov were hydrted for bout one minute fter receiving the semen nd put into 60-liter incubtors, in constntly renewed wter, t 5 l/min. After nine hours of incubtion, the fertiliztion index ws recorded, t gstrul stge (25,5±,5C ). The htching rte rnged from 16 to 18 hours fter beginning the incubtion t room temperture. In order to clculte the fertiliztion percentge nd the htching rte, siphon removed three smples from the incubtor nd 100 eggs were counted in 30X stereoscope. Considering the fertiliztion rte, the embryos tht hd gstrul formtion were considered vible. Concerning the htching rte, lrve moving inside the eggs minutes before htching were considered vible. Three smple verges were tken in three scores. The experimentl design ws completely rndomized. The tretments were rrnged in 3X3 fctor (three hormones in mles nd three hormones in femles). The experimentl unit ws the junction of mle nd femle gmetes. Fertiliztion nd htching vribles were nlyzed ccording to sttistic model, considering the hormones dministered in mle nd femle nimls: Yij = µ + Mi + Fj + MFij + Sk + εij Yijk = observtion of hormones (i) in mle with hormone (j) in femle, (k) in the week; µ = generl constnt; Mi = hormone (i) in mle; Fj = hormone (j) in femle; MFij = mle nd femle hormones interction; Sk = week effect (k); εijk = rndom mistke ssocited to the hormone (i) in mle with hormone (j) in femle, (k) in the week. The sttistic nlysis ws mde using the SAS (1992) progrm. The weight vribles (mle nd femle), sperm concentrtion, semen volume, number of totl sperm, ATU (femle), verge number of spwning oocytes/g, fertiliztion, nd htching rtes were nlyzed through GLM procedure, pplying Tukey test (P<0.05). To the progressive motility nd spermtic vigor vribles, Gmm distribution with function of Log link nd GENMOD nlysis procedure were pplied; to the morphopthologicl vribles, distribution ws Gmm with function of power inverse link nd GENMOD procedure. At lst, x2 (Squre Qui) (P<0.05) ws used in the spwning rte. Results nd discussion Production nd nlysis of P. mesopotmicus semen The verges of semen volume, sperm concentrtion, totl spermtozoon number/relese semen, progressive motility nd spermtic vigor obtined from different gondl inductors re shown in Tble 2. The verge weight of the nimls used in the tretment did not vry (P>0.05); thus, it did not interfere with the prmeters. Tble 2. Animl verge weight, crp, rbbit nd broiler chicken pituitry extrcts effects on semen volume, spermtic concentrtion, totl number of spermtozo number/relesed semen, progressive motility, spermtic vigor, fertiliztion nd htching rtes ccording to semen origin in P. mesopotmicus mle reproducers. Prmeters Pituitry Extrct Broiler chicken Rbbit Crp Animl verge weight 2.55± ± ±0.3 (kg) Semen volume 11.2± ±0.7b 12.0±2.7 (ml) Spermtozo concentrtion 21.1±1,2b 31.0±3,2 20.5±2,3b (mm 3 ) 1 Totl spermtozoon 23.9±2,1 1.13±0,5b 25.1±3,1 number/relese semen 2 Progressive motility 72.8± ± ±7.3 (%) Spermtic vigor 2.71± ± ±0.2 (0 to 5 score) Fertiliztion ccording to 52.7± ± ±2.2 semen origin (%) Htchery ccording to semen origin (%) 50.9± ± ±2.5 Different letters on the sme row (P<0.05); 1 number x10 6 ; 2 number x The verge of semen volume (11.2 ml) produced by the P. mesopotmicus induced with extrct of broiler chicken s pituitry (BCPE) did not differ (P<0.05) from the ones induced with of crp pituitry extrct (CPE) (12 ml). However, the P. mesopotmicus induced with rbbit pituitry extrct (RPE) resulted in the lowest semen volume (1.8 ml) (Tble 2). The explntion for the reduced semen volume of fishes induced with rbbit extrct pituitry could be relted to the rbbits' ge (60 to 70 dys old) when bted. When the pituitries were collected, they might hve contined low FSH nd LH rtes becuse of sexul immturity. Under norml breeding conditions, rbbits cn rech puberty in three or four months, which is considered norml time for niml's libertion of gmetes nd mnifesttion of sexul behvior (Hfez nd Hfez, 2000). Animl pituitry extrct between 60 nd 70 dys ws used becuse this is the ge for commercil

5 Pituitry extrct on Pirctus mesopotmicus 443 slughter. On the other hnd, the birds were slughtered before the gondl prime mturity, but the pituitry responded positively. The concentrtion of spermtozo/mm 3 ws lower (P<0.05) in nimls treted with CPE nd BCPE when compred to nimls treted with RPE (Tble 2). The highest spermtic concentrtion observed in fish treted with RPE my be relted to the lowest volume observed in fish treted with CPE nd BCPE. From this perspective, the results observed by Silveir et l. (1990) were considered, working with P. mesopotmicus induced by hcg, obtining n verge volume of 5.02 ml nd sperm concentrtion of bout 28x10 6 /mm 3. These results re equl to those observed in the nimls induced with RPE, where men semen volume of 1.8 ml nd men concentrtion of 31x10 6 were obtined. Ginzburg (1972) reported the sperm concentrtion in mm 3 in mny species; he observed mens of bout 2.51x10 6 in sturgeon (Huso huso), possibly reching 52x10 6 in perch (Perc fluvitilis), or 73x10 6 in Cspilos kessleri. The verge semen concentrtion sperm/mm 3 found in this study for fish treted with BCPE ws 21x10 6. It suggests tht induced ction in sperm production, ccording to literture, hs good concentrtion ssocited with semen volume nd my ssure efficient oocyte fertiliztion. The totl spermtozo s number/relesed semen is directly relted to volume nd concentrtion. The totl sperm number seems to be lower (P<0.05) in nimls induced by RPE when compred to the ones induced by BCPE nd CPE (Tble 2). To ssure the fertiliztion of ll oocytes, it is necessry to obtin good production of semen with good concentrtion, in order to chieve sufficient sperm quntity. In this study with P. mesopotmicus, n increse of totl cell production ws observed in the control (CPE). Equl increse ws observed in the nimls treted with BCPE, compring to Bedore (1999), who induced the nimls with CPE, nd lso compring them with non-induced nimls. The uthor observed tht the induced nimls produced totl of 26.3x10 10 sperm, but the totl production obtined in controls ws of 5.7x10 10 sperm, where n increse of the totl cell number ws observed. The sme resercher observed identicl results when inducing the pircnjub (Brycon orbignynus), where nimls treted with CPE produced totl sperm number higher thn nimls not induced (14x10 10 nd 8.6x10 10, respectively). Prdo-Crrsco (2001) studied the qulity of ymú semen (Brycon siebenthle) induced with CPE, GnRH- (nlogue mmmlin gondotropin relesing hormone) nd verified tht nimls treted with CPE produced ten times more semen when compred to the controls, but there ws no increse in totl sperm number. There ws just more fluidity in semen, t lower concentrtion. When pcu s (P. mesopotmicus) spermtic motility nd spermtic vigor were nlyzed, there ws no difference (P>0.05) mong the three pituitry extrct tretments used. Progressive motility nd spermtic vigor do not seem to be ffected by the origin of the pituitry extrct used, but rther by other fctors, such s species, niml s ge, sperm concentrtion nd minly on wter physicl nd chemicl chrcteristics (Ginzburg, 1972). In some species, progressive motility my be ffected by the inducer hormone, s verified by Kuchrczyk et l. (1997), who induced the brem (Abrmis brm) to reproduction using brem nd crp pituitry extrct nd hcg in two doses. The verge volume of semen relesed ws 3.5 to 4.0 ml, with progressive motility of 80%, with the control group verge semen volume being 1.6 ml, with motility of 22.8%. Pituitry extrct dministrtion in P. mesopotmicus seems to increse semen volume nd, therefore, the totl sperm number, but it did not ffect progressive motility or spermtic vigor. Fertiliztion between oocytes nd semen under different tretments did not differ (P>0.05), with the sme result observed in the htching rte (Tble 2). It is importnt to remrk tht there ws tendency towrds lower verges of fertiliztion nd htching rtes in the ov fertilized with semen from mle nimls treted with RPE, when compred to the ones treted with CPE nd BCPE. Coser et l. (1992) evluted the curimtá-pcu s (Prochilodus mrggrvii) semen cpcity of fertiliztion, using CPE, nd obtined vlues of 96.4%. Lhnsteiner et l. (1998) emphsized tht, in rinbow trout (Oncorhynchus mykiss), good qulity semen should show progressive motility over 75%, fertilizing, therefore, 80% of ov. In this study, with P. mesopotmicus, the verge of progressive sperm motility ws observed in ll tretments nd vried from 70 to 80%, but fertiliztion did not exceed the verge of 57%. The fertiliztion my hve been ffected not only by oocytes qulity, but by different fctors, such s high quntity of bnorml sperm, for exmple. The motility, volume nd vigor of nimls treted with BCPE nd CPE were the sme nd probbly did not influence fertiliztion. In mmmls, fertiliztion rte is ordinrily relted to the percentge of bnorml sperm, ccording to Hfez nd Hfez (2000), who ssert tht pthology rtes over 20% cn reduce the fertiliztion rte in

6 444 Streit Júnior et l. femle nimls. When the sperm morphology ws studied, normlity rtes of 62% were verified in the mle nimls treted with BCPE nd 63% for CPE (P>0.05), but 50.6% for RPE (P<0.05). When sperm pthologies considered secondry, such s shoehook, tilless nd hedless, were nlyzed, higher percentge (P<0.05) ws observed in P. mesopotmicus induced with RPE (35.9%), compred to the ones induced with BCPE (25.9%) nd with CPE (24.6%). Regrding to more common primry pthologies, such s bent til, coiled til, crooked til nd smll hed, the results were identicl (P>0.05) in the three tretments (Figure 1), with the following percentges: 12.1% for BCPE induction, 12.4% for CPE induction, nd 13.5% for RPE. spermtozo (%) b NS PSP SSP b CPE BCPE RPE Figure 1. Influence tretments of P. mesopotmicus to crp pituitry extrct (CPE), broiler chicken pituitry extrct (BCPE) nd rbbit pituitry extrct (RPE) in norml spermtozo (NS) nd primry spermtozo pthology (PSP) nd secondry spermtozo pthology (SSP). In this experiment, the secondry nd primry pthologies reched n verge of 37.5% in nimls induced with BCPE nd control (CPE) nd of 49.4% in the ones induced with RPE. These vlues cn be considered high when compred with mmmls. The hypotheses rised for tht sitution my relte to indequte diet of reproductive nimls, dvnced or rther erly ge, nd consnguinity. The dt obtined from semen of nimls induced with RPE were interesting, the rte of primry spermtic pthologies being similr to tht of nimls treted with BCPE nd CPE. It ws not possible, however, to nlyze whether the rtes of pthologies re relted to the inducers, which my cuse chnges during the spermtozo formtion. This kind of informtion is importnt to verify pthologies influence on oocyte fertiliztion rtes, s in the mmml's cse (Colégio Brsileiro de Reprodução Animl, 1998), lthough Nghm (1983) concluded tht there re few differences between mmml nd fish spermtogenesis. Evlution of P. mesopotmicus spwning The spwning of femles with identicl verge weight (P<0.05), under tretment with CPE, BCPE nd RPE ws evluted. Ov weight, oocytes per grm of ov, fertiliztion rte nd htching rte were evluted in the CPE nd BCPE tretments, but it ws not possible to evlute femles treted with RPE, since there ws no response from them. Dt re in Tble 3. Tble 3. Averge niml weight nd tretment effects with crp, broiler chicken nd rbbit hypophysis extrct on P. mesopotmicus spwning induction, ccumulted therml unit, verge number of oocytes/g of spwning. Prmeters Animls weight verge (Kg) Spwning rte (%) Accumulted Therml Unit (TAU) Averge oocytes number/g of spwning Fertiliztion rte (%) Htchery rte (%) Different letters on the sme row (P<0.05). Pituitry Extrct Broiler Rbbit Crp Chicken 2.7± ± ± ±20 0b 83.0± ± ± ± ± ± ± ± ±9.9 The number of P. mesopotmicus tht spwned while induced by BCPE ws similr to those induced by CPE (P>0.05), but the nimls treted with RPE did not rect stisfctorily to the spwn (Tble 3). This lck of response is probbly due to the sme hypotheticl resons lredy mentioned for mles, such s the precocious ge of the rbbits tht supplied the pituitry or the incomptibility of the rbbit s gondotrophins dministered to fish. The BCPE proved to be efficient in more thn 70% of the induced femles. The high percentge ttests its potentility when compred to the ction of other hormones, s described in Romgos et l. (1990), who induced P. mesopotmicus femles using hcg nd slmon pituitry extrct, obtining 57.9 nd 84% of efficcy, respectively. Amrl Jr. et l. (1996) obtined 90%, employing crp extrct on Cyprinus crpio, Silv et l. (1997) obtined 100% of response working with BCPE in curimbtás. The high vribility of numbers is due to differences mong species nd to the mteril used for induction. Amrl Jr. (1995) stted tht vin pituitry hormones re chemiclly similr to teleosts hormones. In ddition, the minocids sequences of the hypothlmic hormones re very similr mong fish nd birds. According to Donldson (1996), there re two or three distinct forms of GnRH in vrious fish species. Yu et l. (1995) tested pituitry

7 Pituitry extrct on Pirctus mesopotmicus 445 extrct of broiler chicken, duck, turkey, ostrich, nd goose on the spwning of Prmisgurnus dbrynus, Misgurnus nguillicudutus, nd Hypophthlmichthys molitrix. They reported good results from the first two inducers, in which lbortoril tests indicted the FSH presence, which certinly cts s GTH (gondotrophin hormone) on the stimultion of estrdiol-17 (in vitro) production. As Brroso (1999) stted, the P. mesopotmicus hd positive rection to BCPE. This study showed good results for BCPE s gondl inducer, promoting spwn to stisfctory results nd showing promising perspectives for BCPE. No difference (P>0.05) concerning the vrible ATU ws verified between CPE nd BCPE tretments (Tble 3), lthough tendency for higher verge in nimls induced with BCPE ws noticed. In gret prt of the experiment, it ws observed tht nimls induced by BCPE took longer to spwn, reching 291 ATU, wheres the nimls treted with CPE did not exceed 263 ATU. This ATU difference between CPE nd BCPE tretments, in wter t the sme temperture, llows the supposition tht BCPE gondotropins tke longer to be bsorbed, enter the bloodstrem nd ct in the gonds of femle P. mesopotmicus. Godinho nd Godinho (1986) induced the sme species to spwning by dministering CPE nd observed n ATU of 325 nd 289 fter the second injection t temperture tht rnged from 22 to 24.5 C. The ATU results of the CPE experiment were resonbly lower, due to the verge temperture of 25.5 C. Among other fctors, the ATU vlue depends minly on the wter temperture in the tnk. This sttement is reinforced by Lm (1983), who sid tht temperture ws the most importnt fctor t spwning control. The ATU vries ccording to the species nd the inducers used, s in Lee et l. (1988) experiments, where the uthors reported 328 of ATU in the mullet (Mugil cephlus), using CPE nd ATU, with LhRH- (nlog Luteinizing Relesed Hormones). Kuchrczyk et l. (1998) reported ATU of 230 to 348, from the ppliction of the second dose until the spwning, when studying the use of mixture of FSH+LH ssocited with pimozide or methocloprmide (Sigm Chemicl Co., St. Louis, MO, USA) for spwning nd sperm production of perch (Perc fluvitilis). During this study, it ws observed tht it took longer for the nimls induced with BCPE to spwn, reching up to 291 ATU; wheres this vlue did not go over 263 ATU for the ones treted with CPE. The verge number of oocytes per grm of ov did not differ (P>0.05) from the nimls treted with CPE nd with BCPE (Tble 3). Ceccrelli et l. (2000) stted tht the expected number of oocytes per grm of ov for P. mesopotmicus is In this experiment, the number ws, in verge, 20% higher. This number vries ccording to the species, s verified by Sto et l. (1996) who treted white piu (Schizodon knerii) with pituitry extrcts nd obtined 930 oocytes per grm of ov. This vrible is directly linked to the niml size nd its species (Ceccrelli et l., 2000). However, Lovell (1989), who studied cnl ctfish femles (Ictlurus puncttus), stted tht reproductive chrcteristics such s: number of eggs, egg size, spwning rte, nd lrve qulity re directly relted to nutritionl condition. Such informtion my led to the belief tht pituitry extrct does not ffect the number oocytes/grm of spwning, since in this study the vlues were identicl in ll tretments, lthough there were no non-induced nimls to compre with. Fertiliztion nd htching rte from CPE nd BCPE tretments proved to be similr (P>0.05) (Tble 3). Fertiliztion rte nd htching rtes might be directly linked to feeding. Wtnbe (1985) ffirmed tht n dequte diet for breeders might improve spwning qulity s well s the produced eggs, resulting in helthy, good-qulity fingerlings. Brzusk nd Grzywczewski (1999) induced crps (Cyprinus crpio) from two different lineges, using CPE nd pelletized GnRH, obtining fertiliztion index of 93%. Bernrdino et l. (1993) induced the mtrinchã (Brycon cephlus) using CPE nd observed n verge of 85% in fertiliztion. Sto et l. (1996) lso employed CPE on white piu (Scizodon knerii) to induce spwning obtining 69% of fertiliztion. Rezende et l. (1996) induced the Leporinus elongtus resulting in 73% fertiliztion rte. This study showed low fertiliztion rtes (57% nd 49.6%) nd htching rtes (57% nd 46.9%) for nimls induced by CPE nd BCPE, respectively. These figures my suggest tht the origin of the pituitry extrct ws determining effect on these rtes. On the other hnd, fctors enumerted by Bromge (1995) such s the qulity of breeders feeding, environmentl conditions, stress t cpture, mnipultion in the induction tnk, genetics, incubtion conditions nd mturity, mong others, my ffect the rtes.

8 446 Streit Júnior et l. Conclusion The results of this study show tht broiler chicken pituitry extrct, when compred to crp pituitry extrct, proved to be effective in inducing spwning nd sperm production. On the other hnd, rbbit pituitry extrct did not respond positively s good gonodl inducer on mles nd femles P. mesopotmicus. Semen nlysis from nimls treted with broiler chicken pituitry extrct nd crp pituitry extrct showed similr qulity. According to these results, new studies re recommended, especilly the ones relted to the ge of rbbit pituitry extrct. Acknowledgements Authors thnk Ntionl Council Scientific nd Technologicl Reserch (CNPq) by the finncil support. References AMARAL JÚNIOR, H. Utilizção de extrto hipofisário de glinh pr indução desov de tenc. Opção de bnco de hipófise pr o pequeno produtor rurl. In: ENCONTRO RIOGRANDENSE DE TÉCNICOS EM AQÜICULTURA, 6., 1995, Ibirubá. Anis... Ibirubá: UFRGS, 1995, p AMARAL JUNIOR, H. et l. Observções preliminres d reprodução semi-rtificil de crp comum, Cyprinus crpio, utilizndo indução hormonl e mbiente controldo, pr desov nturl em substrto tipo ráfi. In: SIMPÓSIO BRASILEIRO DE AQUICULTURA, 9., 1996, Sete Lgos. Anis... Sete Lgos: ABRAQ, 1996, p. 64. BARROSO, R.M. Utilizção do extrto bruto de hipófise de frngo de corte (Gllus domesticus) n induçãor d mturção finl oocitári e d desov em pcu (Pirctus mesopotmicus) (Holmberg, 1887) Dissertção (Mestrdo)-Centro de Ciêncis Médics, Universidde Federl Fluminense, Rio de Jneiro, BARTH, A.D.; OKO, R.J. Abnorml morphology of bovine spermtozo. Ames: Iow Stte University Press, BEDORE, A.G. Crcterístics e criopreservção do sêmen de pcu-crnh (Pirctus mesopotmicus) e de pircnjub (Brycon orbigninus) Dissertção (Mestrdo) Instituto de Ciêncis Biológics, Universidde Federl de Mins Geris, Belo Horizonte, BERNARDINO, G. et l. Propgção rtificil do mtrinchã, Brycon cephlus (Guenther,1869), (Teleostei, Chrcide). Boletim Técnico do CEPTA, Pirssunung, v. 6, n. 2, p. 1 9, BROMAGE, N. Broodstock mngement nd seed qulity-generl considertions. In: BROMAGE, N.; ROBERTS, R.J. (Ed.). Broodstock mngement nd egg lrvl qulity. Oxford: Blckwell Science, cp. 1, p BRZUSKA, E.; GRZYWACZEWSKI, R. Artificil spwning of crp Cyprinus crpio L.: differences between the effects on reproduction in femles of Isreli strin Dor-70 nd its cross-breed treted with crp pituitry nd Ovopel. Aqucult. Res., Oxford, v. 30, n. 8, p , CASTAGNOLLI, N. Espécies exótics própris pr piscicultur. In: CASTAGNOLLI, N. (Ed.). Piscicultur de águ doce. Jboticbl: FUNEP, cp. 2, p CASTAGNOLLI, N.; DONALDSON, M.E. Induced ovultion nd rering of the pcu (Colossom mitrei). Aquculture, Amsterdm, v. 25, n. 2, p , CECCARELLI, P.S. et l. Dics em piscicultur; pergunts e resposts. Sntin Gráfic Editor: Botuctu, CBRA-COLÉGIO BRASILEIRO DE REPRODUÇÃO ANIMAL. Mnul pr exmes ndrológicos e vlição de sêmen niml. Belo Horizonte: CBRA, CONN, H.J. The microscopic study of bcteri nd fungi in soil. Technol. Bull., Atlnt, v. 64, n. 1, p. 61, COSER, A.M.L. et l. Cpcidde de fertilizção do sêmen de Prochilodus mrggrvvi (curimtã-pcu) congeldo sob form de pellets. In: ENCONTRO ANUAL DE AQÜICULTURA DE MINAS GERAIS, 10., 1992, Belo Horizonte. Anis... Belo Horizonte: AMA, 1992, p DONALDSON, E.M. Mnipultion of reproduction in frmed fish. Anim. Reprod. Sci., London, v. 42, n. 1-4, p , DONALDSON, E.M., HUNTER, G.A. Induced finl mturtion, ovultion, nd spermition. In: HOAR, W.S. et l. (Ed.). Fish physiology. (v.9b). Orlndo: Acdemic Press, cp. 7, p GINZBURG, A.S. The spermtozoon. In: DETLAF, T.A. (Ed.). Fertiliztion in fishes nd the problem of polyspermy. Jeruslém: Keter Press, cp. 2, p GODINHO, H.P.; GODINHO, A.L. Induced spwning of the pcu Colossom mitrei (Berg 1895), by hipophystion with crude crp pituitry extrct. Aquculture, Amsterdm, v. 55, n. 1, p , GODINHO, H.P. et l. Sobre reprodução induzid de pcu, Colossom mitrei (Berg, 1985). Cienc. Cult., Cmpins, v. 29, n.(especil), p , HAFEZ, E.S.E; HAFEZ, B. Reproduction in frm nimls. Phildelphi: Lippicott Willims e Wickins, HANCOCK, J.L. Stinig technique for the study of temperture shock in semen. Nture, London, v. 167, n. 4243, p , KUCHARCZYK, D. et l. Induced spwning in brem, Abrmis brm (L.), using crp nd brem pituitry extrct nd HCG. Aqucult. Res., Oxford, v. 28, n. 2, p , KUCHARCZYK, D. et l. Induced spwning in perch, Percfluvitilis L., using FSH+LH the pimozide or metocloprmide. Aqucult. Res., Oxford, v. 29, n. 1, p , LAHNSTEINER, F. et l. Determintion of semen qulity of the rinbow trout, Oncorhinchus mykiss, by sperm motility, seminl plsm prmeters, nd spermtozo metbolism. Aquculture, Amsterdm, v. 163, n. 1, p , 1998.

9 Pituitry extrct on Pirctus mesopotmicus 447 LAM, T.J. Environmentl influences on gondl ctivity in fish. In: HOAR, W.S., et l. (Ed.). Fish physiology. (v.9b) Orlndo: Acdemic Press, cp. 2, p LEE, C.S. et l. The cost nd effectiveness of CPH, hcg nd LHRH- on the induced spwning of grey mullet, Mugil cephlus. Aquculture, Amsterdm, v.73, n. 2, p , LOVELL, T. The concept of feeding fish. In: LOVELL,T. (Ed.). Nutrition nd feeding of fish, New York: Vn Nostrnd Reinhold, cp. 1, p NAGAHAMA, Y. The functionl morphology of teleost gonds. In: HOAR, W.S. et l. (Ed.). Fish physiology. (v.10). Orlndo: Acdemic Press, cp. 5, p PARDO-CARRASCO, S.C. Reprodução induzid do ymú, Brycon siebenthle (Pisces Chrciforme) Tese (Doutordo)-Centro de Ciêncis Agráris, Universidde Federl de Snt Ctrin, Florinópolis, REZENDE, A.P.S. et l. Avlição d mturção gôndl e indução d reprodução de peixes coletdos no Rio Grnde, jusnte de UHE Ituiuting, MG. Arq. Brs. Med. Vet. Zootec., Belo Horizonte, v. 48, n. 1, p , ROMAGOSA, E. et l. Reprodução induzid do pcu Colossom mitrei Berg, 1895, mntidos em condições de cultivo intensivo. Cienc. Cult., Cmpins, v. 37, n. 7 (supl.), p. 849, ROMAGOSA, E. et l. Pttern of oocyte dimeter frequency distribution in femle of the pcu, Pirctus mesopotmicus (Holmberg 1887) (= Colossom mitrei Berg 1895), induced to spwn. Aquculture, Amsterdm, v. 86, n. 1, p , SAS Institute Inc. SAS technicl report. Relese 6.07 Cry: NC, SATO, Y. et l. Hypophystion of the nostomid fish white-piu Schizodon knerii from the Rio São Frncisco bsin. Arq. Brs. Med. Vet. Zootec., Belo Horizonte, v. 48, n. 1, p , SILVA, J.A. et l. Utilizção de extrto cru de hipófise de frngos (Gllus domesticus) como indutor de desov em Curimtá (Prochilodus scrof). Rev. Brs. Reprod. Anim., Belo Horizonte, v. 21, n. 2, p , SILVEIRA, W.F. et l. Avlição espermátic, preservção criogênic e fertilidde do sêmen do pcu, Pirctus mesopotmicus (Holmberg), proveniente de reprodução induzid. Bol. Inst. Pesc, São Pulo, v. 17, n. 1, p. 1-13, SORENSEN Jr., A.M. A lbortory for niml reproduction. Americn Press: Msschusetts, YU, J.Y.L. et l. Comprtive effects of vin nd piscine gondotrophins on gondl steroidogenesis, nd of vin nd piscine pituitries on induction of spermition nd ovultion in the loch nd white silver crp. Aquculture, Amsterdm, v. 135, n. 1, p , WATANABE, T. Importnce of the study of broodstock nutrition or further development of quculture. In: COWEY, C.B. et l. (Ed.). Nutrition nd feeding nd fish, London: Acdemic Press, cp. 8, p WOYNAROVICH, E.; HORVÁTH, L. A propgção rtificil de peixes de águs tropicis: mnul de extensão. Brsíli: Escopo, Received on Mrch 03, Accepted on November 21, 2005.