Zygote 21 (February), pp. 8594. C Cambridge University Press 2011doi:10.1017/S0967199411000396 First Published Online 15 August 2011

Structural analysis of oocytes, post-fertilization events andembryonic development of the Brazilian endangered teleostBrycon insignis (Characiformes)

Ziara A. Isa2, Elizete Rizzo

3, Thiciana B. Amaral

2, Natlia M.N. Mourad

2and Ana T.M. Viveiros

1

Dept of Animal Science (DZO), Federal University of Lavras (UFLA); and Institute of Biological Science (ICB), FederalUniversity of Minas Gerais (UFMG), MG, Brazil

Date submitted: 30.11.2010. Date accepted: 28.05.2011

Summary

The aim of this study was to evaluate the oocytes, post-fertilization events and embryonic developmentin Brycon insignis, under both scanning electronmicroscopy and stereomicroscopy. Oocytes and embryoswere sampled from spawning up to hatching. Stripped oocytes were spherical, non-adhesive, greenish-brown, possessed a single micropyle, pore-canals and had a mean diameter of 1.46 mm. In 63% ofoocytes the germinal vesicle was peripheric. The main post-fertilization events were the fertilizationcone formation (20 s), micropyle closure (100180 s) and agglutination of supernumerary spermatozoa(100180 s). Embryonic development lasted 30 h at 24 C and was characterized by seven stages.Zygote, cleavage, blastula and gastrula stages were first observed at 0.25, 1, 3 and 6 h post-fertilization,respectively. Fertilization rate was determined at the moment of blastopore closure, 1011 h post-fertilization. The segmentation stage began at 11 h post-fertilization and comprised the development ofsomites, notochord, optic, otic and Kupffers vesicles, neural tube, primitive intestine, and developmentand release of the tail. The larval stage began 21 h post-fertilization and was characterized by thepresence of somites, growth and elongation of the larvae. At the hatching stage, embryos presentedvigorous contractions of the tail and body leading to chorion rupture (30 h). The morphologicalcharacteristics described for B. insignis were similar to that described for other teleost species, and suchknowledge is important for a better understanding of reproductive features of a species and useful forecological and conservational studies.

Keywords: Brycon, Characidae, Egg, Embryogenesis, Fertilization

Introduction

The Brycon insignis (Steindachner, 1877), known astiete tetra in English and piabanha in Portuguese, is anative and endemic species of the Paraba do Sul Riverbasin located at So Paulo, Minas Gerais and Rio deJaneiro States, Southeast Brazil (Hilsdorf & Petrere Jr,2002). The genus Brycon belongs to the order Chara-

1All correspondence to: Ana T M Viveiros. Dept Zootecnia,Universidade Federal de Lavras (UFLA), caixa postal3037, Lavras, MG, 37200000, Brazil. Tel/Fax: +5535 38291231. e-mail: anatmviveiros@hotmail.com orana.viveiros@dzo.ufla.br2Dept of Animal Science (DZO), Federal University of Lavras(UFLA), MG, Brazil.3Institute of Biological Science (ICB), Federal University ofMinas Gerais (UFMG), MG, Brazil.

ciformes, family Characidae, subfamily Bryconinaeand comprises more than 70 fish species with widegeographic distribution (Froese & Pauly, 2010). ManyBrazilian fsh species, B. insignis migrate up-stream tospawn. This migratory behaviour is known as piracemaand lasts from October to February. In the 1950s,B. insignis was the fourth most captured speciesin commercial fishing in this basin, therefore dis-playing an important role in the regions economy(Machado & Abreu, 1952). However, due to over-fshing, pollution and changes in the Paraba doSul River course during the construction of theHydroelectric Paraibuna-Paraitinga, B. insignis re-productive cycle was disrupted and its status iscurrently set as endangered (Andrade-Talmelli et al.,2001).Fertilization is a process of cellular fusion that

encompasses the contact between the spermatozoon

86 Isa et al.

and the oocyte up to the union of the nuclei ofboth cells (Schatten, 1999; Moore, 2001). Fertilizationpromotes the activation of female gamete with theresumption of meiosis, formerly stuck at metaphaseII and triggers a chain of events inside the oocytes(Brasil et al., 2002). As fertilization in fish is generallymonospermic (Kobayashi & Yamamoto, 1981), afterthe first spermatozoon penetration, a series of eventsthat prevents polyspermy takes place. These eventsinclude mechanical barriers such as: (a) closure ofthe micropyle (Kobayashi & Yamamoto, 1981; Hart& Donovan, 1983, Kudo et al., 1994); (b) formationof the fertilization cone (Kudo, 1980; Iwamatsuet al., 1991; Linhart & Kudo, 1997; Brasil et al.,2002; Murata, 2003); and (c) activation of the corticalreaction to eliminate any supernumerary spermatozoa(Iwamatsu & Ohta, 1981; Iwamatsu et al., 1993). Aftercellular fusion, one diploid cell undergoes a processnamed embryonic development or embryogenesis.Knowledge of the embryonic development in fishspecies is useful to the management of fisheryresources and to surveys related to fish culture as itprovides additional information regarding species lifecycles, for ontogeny studies, experimental modeling,evaluation of environmental quality and effects of toxicsubstances on aquatic fauna (Flores et al., 2002), as wellas for experiments on ex situ species preservation.Considering the great number of species, post-

fertilization events and embryonic developmentstudies on neotropical fsh species have receivedlittle attention. Reports on fertilization events andembryonic development in species of the orderCharaciformes are available for matrinx Bryconcephalus (Lopes et al., 1995; Romagosa et al., 2001;Alexandre et al., 2010), pirapitinga Brycon nattereri(Maria, 2008), piracanjuba Brycon orbignyanus (Ganeco,2003; Reynalte-Tataje et al., 2004; Ganeco et al., 2009),jatuarana Brycon spp. (Neumann et al., 2007), aimaraHoplerythrinus unitaeniatus, traro Hoplias lacerdaeand trahira Hoplias malabaricus (Gomes et al., 2007),dorado Salminus brasiliensis (Nakaghi et al., 2006)and streaked prochilod Prochilodus lineatus (Brasil etal., 2002; Ninhaus-Silveira et al., 2006). Preliminaryobservations of some embryonic (cleavage, gastrula,neurula and embryo) and larval development stagesof the B. insignis under stereomicroscopy werereported by Andrade-Talmelli et al. (2001). Oocytes,post-fertilization events, and the zygote and blastulastages of B. insignis however, have not yet beenpublished. The use of scanning electron microscopy(SEM) to monitor morphological changes of oocytesand eggs allows a detailed observation of the externalstructures, provides a better understanding of thefertilization and embryonic development of a fishspecies, and completes the information gatheredunder histologic and stereomicroscopic analysis.

Thus, the aim of the present study was to evaluatethe oocytes, post-fertilization events and embryonicdevelopment in Brycon insignis, under both SEM andstereomicroscopy.

Materials and methods

Fish, gamete collection and initial gamete evaluation

All fish were handled in compliance with publishedguidelines for animal experimentation (Van Zutphenet al., 1993). Tiete tetra Brycon insignis broodfish wasselected from earthen ponds at the Hydrobiology andAquaculture Station of Hydroelectric Company of SoPaulo (CESP) in Paraibuna (232310S; 453944W),So Paulo state, Brazil, during the spawning season(January and February).Two males of 45 years of age (296 28 g)

with detectable running sperm under soft abdominalpressure were given a single intramuscular dose ofcarp pituitary extract (Argent Chemical Laboratory)at 3 mg/kg body weight and maintained at 24 C.Eight hours later, the urogenital papilla was carefullydried, and sperm was hand-stripped directly intotest tubes. Sperm collection was carried out at roomtemperature (2325 C) and, soon after collection, thetubes containing sperm were placed in a Styrofoambox containing crushed ice (5 2 C). Contaminationof sperm with water, urine or feces was carefullyavoided. An aliquot of 5 l of each sample wasplaced on a glass slide and observed under lightmicroscope (Model L1000, Bioval, Jiangbei, China)at 400 magnification. As fish sperm in seminalplasma should be immotile, any sperm motility wasconsidered to have undergone premature inductionresulting from urine or water contamination and thesample was discarded. In samples thusly selected,sperm motility was triggered in 25 l of 1% NaHCO3(Labsynth) as activating agent (Viveiros et al., 2011) andsubjectively estimated under light microscope. Spermconcentration (hemacytometer, Neubauer chamber)was also determined.To harvest oocytes, eight females of 12 years of

age (814 297 g) with detectable running oocytesunder soft abdominal pressure, received two dosesof carp pituitary extract (0.5 and 4.5 mg/kg bodyweight) at 12 h interval. Together with the seconddose, females received a dose of human chorionicgonadotropin (PregnylTM Schering-Plough) at 1500 IUhCG/kg body weight and were hand-stripped 8 hlater, at24 C. The spawning weight, spawning index(spawning weight 100/body weight), number ofoocytes/g ova and the number of oocytes/female werecalculated. Three aliquots of oocytes from each femalewere collected. The first aliquot (n = 30 oocytes

Oocytes and embryos in Brycon insignis 87

8 females) was fixed in Gilsons solution (50 ml 60%ethanol, 440 ml distilled water, 7 ml nitric acid, 10 gmercuric chloride, 9 ml glacial acetic) and the diameterof each oocyte was measured under a light microscopeusing a micrometric objective. The second aliquot (n= 30 oocytes 8 females) was fixed in Serrassolution (60 ml 90% ethanol, 30 ml 37% formaldehyde,10 ml glacial acetic) and the germinal vesicle position(central, peripheric and breakdown) was examinedunder a stereomicroscope (TIM 2 T, Opton). Finally,the third aliquot (n = 20 oocytes 8 females) wasfixed in modified Karnovsky (2.5% glutaraldehyde,2.5% paraformaldehyde in 50 mM sodium cacodylatebuffer, pH 7.2, 1 mM CaCl2) and transported to theLaboratory of ElectronMicroscopy and UltrastructuralAnalysis at Federal University of Lavras (UFLA), inLavras, Minas Gerais State, Brazil. Then oocytes werepost-fixed in 1% osmium tetroxide for 4 h at roomtemperature, washed in 0.1 M cacodylate buffer (pH7.4), dehydrated through grade acetone solutions (25,50, 75, 90 and 100%), dried with CO2 in a PELCOCPD 030 critical point, coated with gold under vacuumconditions with SEM Coating Unit SCD 050 andexamined with a scanning electron microscope (LEOEVO 40 XVP ESC Carl Zeiss, Santo Amaro, SP, Brazil)equipped with a digital camera.

Fertilization process

Freshly collected sperm was used to fertilize oocytesin a factorial of two males two females, performinga total of four progenies. An approximate ratio of 5.0105 1 105 spermatozoa: oocyte was used, based onour previous study with another species of the genusBrycon (Maria et al., 2006). To achieve that ratio, 5 gof oocytes (2835 oocytes) was fertilized with 60 lof sperm. Fertilization was initiated by the addition of10 ml tank water and agitated for 1 min. Subsequently,20 ml tank water was added and samples mixed foranother 2 min. Finally, eggs were transferred to fourfunnel type incubation units (1 progeny= 1 incubationunit) made with 1.5-litre plastic bottles with 10 cm ofdiameter, and incubated in a fow-through system at24 C.

Post-fertilization events

Approximately 20 eggs from each incubation unit werecollected at 0 (when sperm was mixed with oocytebut before the addition of water), 20, 40, 60, 80, 100,120, 140, 160 and 180 s post-fertilization. Eggs werefixed in Karnovskymodified and then examined underSEM as described for oocytes. The spermatozoa arrivalat the micropyle and the following post-fertilizationevents were tracked: fertilization cone formationwith the expulsion of supernumerary spermato-zoa, agglutination of supernumerary spermatozoa in

the vestibule, and the beginning of the micropyleclosure.

Embryonic development

Approximately 20 eggs from each incubation unitwith apparent normal development were collectedat 0.25, 0.5, 1 and 1.5 h, every 1.5 h until 6 hand every 5 h until hatching. Eggs were fixed in2.5% glutaraldehyde, transported to the Laboratoryof Semen Technology at UFLA, examined understereomicroscopy and photographed with digitalcamera (DSC-W35, Sony Electronic Inc, New York,NY, USA). The following main stages of embryonicdevelopment were tracked: zygote, cleavage, blastula,gastrula (blastopore closure), segmentation, larval andhatching (Ninhaus-Silveira et al., 2006).When most of the developing embryos were

observed at the blastopore closure stage, 100200eggs were randomly collected from each incubationunit and the number of fertilized eggs (transparentwith a developing embryo), as a percentage of totaleggs (transparent + dead or white), was determined.After counting, eggs were placed back into the sameincubation unit for further development.

Results

Initial gamete evaluation

B. insignis fresh sperm possessed a mean of 23.5 109 1.5 109 spermatozoa/ml and 95% motilesperm upon activation. Recently stripped oocyteswere spherical, non-adhesive and greenish-brown.Spawning weight of 125 g, spawning index of 15.2%,567 oocytes/g ova, 70179 oocytes/female and amean oocyte diameter of 1.46 mm were observed(Table 1). The germinal vesicle position was centralin 2% of stripped oocytes, peripheric in 63%, andbreakdown in 35%. Under SEM observation, oocytesurface, which corresponds to the chorion or zonaradiata, possessed a single micropyle and pore-canals(Fig. 1A). Themicropyle was characterized by a conicalvestibule and a narrow micropylar canal (Fig. 1B).At the animal pole, diameter and number of pore-canals increased towards the micropyle (Fig. 1B,C)whereas at the vegetative pole, the pore-canals showeda similar diameter and were uniformly distributed(Fig. 1D).

Post-fertilization events

Somemorphological post-fertilization events observedunder SEM are depicted in Fig. 2AD and Table 2.Several spermatozoa were observed at the entranceof the micropyle canal when sperm was added to

88 Isa et al.

Table 1 Female body weight and oocytes characteristics (n = 8 females) oftiete tetra Brycon insignis after hormone treatment

Characteristics Mean SD Minimummaximum

Female body weight (g) 814 297 4001200Spawning weight (g) 125 52 55178Spawning index (%)a 15.2 2.4 12.519.8Number of oocytes/g ova 567 29 530590Number of oocytes/female 70,179 27,248 32,175100,890Oocyte diameter (mm) 1.46 0.08 1.281.79

aSpawning index: spawning weight 100/body weight.

Figure 1 Scanning electron micrographs of the oocyte surface of tiete tetra Brycon insignis. (A) Single micropyle (arrow) at theoocyte surface. (B) Details of micropyle (m) with vestibule (v), micropylar canal (c) and pore-canals (arrow) at the animal pole.(C) Fractured zona radiata (Zr) with pore-canals (arrow) at the oocyte surface(s). (D) Regular distribution of pore-canals at thevegetative pole.

oocytes before the addition of water (time 0; Fig. 2A)up to 20 s post-fertilization. The fertilization conewas characterized as a spherical structure blocking theentrance of micropyle and was observed 20 to 40 s

post-fertilization (Fig. 2B). Agglutinated supernumer-ary spermatozoa expelled from the micropylar canal(Fig. 2C) and the beginning of micropyle closure wereobserved 100180 s post-fertilization (Fig. 2D).

Oocytes and embryos in Brycon insignis 89

Table 2 The moment when some post-fertilization events were first observed in species of the genus Brycon

Seconds post-fertilization

Events B. insignisa (24 C) B. nattererib (19 C) B. orbignyanusc (27 C) Brycon spp.d (27 C)

Spermatozoa at themicropyle

020 1020 090 1030

Fertilization cone 2040 60 60 From 10Agglutination of

supernumeraryspermatozoa

100180 60240 ND 80

Beginning micropyleclosure

100180 120 ND ND

ND, not determined.aThe present study; bMaria (2008); cGaneco et al. (2009); dNeumann et al. (2007).

Figure 2 Scanning electron micrographs of some post-fertilization events in tiete tetra Brycon insignis. (A) Arrival ofspermatozoa at the micropylar canal, 020 s post-fertilization. (B) Fertilization cone (arrow), 2040 s post-fertilization.(C) Supernumerary spermatozoa expelled from the micropyle, 100180 s post-fertilization. (D) Reduced micropylar canaldiameter 100180 s post-fertilization.

Embryonic development

The embryonic development from fertilization tohatching lasted approximately 30 h at 24 C (720h-degree; Table 3). Under stereomicroscopy ana-lysis, the following embryonic development stageswere identified: zygote, cleavage, blastula, gastrula

(blastopore closure), segmentation, larval and hatching(Fig. 3 and Table 3).The zygote stage was identified at 0.25 h and lasted

up to 1 h post-fertilization (Fig. 3A). This stage werecharacterized by: (a) the formation of the blastodiscon the animal pole comprising the active cytoplasmand nucleus; (b) the formation of the vegetative

90 Isa et al.

Table 3 The moment when the embryonic development stages were first observed in species of the genus Brycon

Hours post-fertilization

StagesB. insignisa

24 CB. insignisb

2527 CB. cephalusc

27 CB. nattererid

19 CB. orbignyanuse

27 C

Zygote 0.25 ND 0.25 1.75 0.3Cleavage 1 0.7 0.5 15 0.5Blastula 3 2.5 1.25 ND 2Gastrula 6 4 1.75 21 3Blastopore closuref 1011 7 6 26 7Segmentation 11 8.5 7 29 8Larval 21 10 9 41 11Hatching 30 14 11 5054 13

ND, not determined.aThe present study; bAndradeTalmelli et al. (2001a); cAlexandre et al. (2010); dMaria (2008); eGaneco (2003).fOr 90% of epiboly.

pole composed of yolk; and (c) the egg hydrationcharacterized by the increase of the egg diameter from1.46 to 4 mm post-fertilization.The cleavage stage (from 1 to 3 h; Fig. 3B) was

characterized by the beginning of cell division into 2,4, 8, 16, 32 and 64 blastomeres. The cleavage followedmeroblastic pattern and blastomeres of distinct sizeswere observed. The cleavage pattern was observed asfollows: the first cleavage plane was vertical, givingrise to two blastomeres; the second plane was verticaland perpendicular to the first one, giving rise tofour blastomeres; the third cleavage was vertical andparallel to the first one, giving rise to eight blastomeresin a 4 2 arrangement; the fourth was vertical andparallel to the second cleavage, giving 16 blastomeresin a 4 4 display; the fifth plane was vertical andparallel to the first cleavage, giving 32 blastomeresin a 4 8 formation; and the sixth cleavage planewas horizontal, giving rise to two cell layers, with atotal of 64 blastomeres. Between 1 and 3 h, embryosin different development stages (zygote, cleavage withdifferent number of blastomeres, and blastula) wereidentified in a given sampling time. From 3 h onwards,embryos developed more uniformly.During the blastula stage (from 3 to 6 h; Fig. 3C),

blastomeres reorganized and blastoderm possessed ahalf-moon shape. The blastomeres underwent continu-ous divisions, but their pattern was undetermined.The gastrula stage (from 6 to 11 h; Fig. 3D)

was characterized by the migration of blastodisc cellsinto the vegetative pole through epiboly movement,with gradual cell expansion around the yolk. Twoembryonic layers were formed at this stage: theepiblast and hypoblast. Epibolymovement culminatedwith blastopore closure when the yolk was completelysurrounded by the blastoderm forming the yolk sac(Fig. 3E). At this moment, a fertilization rate of 26%was calculated.

The segmentation stage (from 11 to 21 h; Fig.3F) began with the differentiation of embryonic germlayers. The somites, notochord, neural tube, earlydelimitation of the intestine and elongation of theembryo (mainly at the headtail axis) were observedat this stage. Approximately 16 h post-fertilization, theembryos possessed about 18 somites, optic, otic andKupffers vesicles at the caudal region and an attachedtail.The larval stage began at 21 h of the development

and was characterized by the presence of a free tailand more than 26 somites. The notochord extendedfrom the cephalic region up to the tail and a welldefined primitive posterior intestine could be observed(Fig. 3G).Vigorous muscle contractions of both the tail and

body lead to chorion rupture and hatching of larvae30 h post-fertilization. Hatched larvae possessed atransparent and elongated body (Fig. 3H).

Discussion

Stripped oocytes of Brycon insigniswere spherical, non-adhesive, greenish-brown, 1.46 mm of diameter andthe germinal vesicle position was peripheric in 63%of the oocytes. These characteristics were similar tothose reported for other species of the genus Brycon(Eckmann, 1984; Vazzoler, 1996; Andrade-Talmelliet al., 2001; Romagosa et al., 2001; Reynalte-Tataje et al.,2004; Ganeco et al., 2009; Alexandre et al., 2010). Theoocyte surface of B. insignis possessed a single funnel-like shaped micropyle and pore-canals, similarly toother species of Characiformes (Rizzo et al., 2002;Ganeco & Nakaghi, 2003; Ninhaus-Silveira et al.,2006; Neumann et al., 2007; Maria, 2008; Ganecoet al., 2009; Alexandre et al., 2010). The micropyleis a concave region located at the oocyte surface,

Oocytes and embryos in Brycon insignis 91

Figure 3 Stages of embryogenesis of tiete tetra B. insignis, observed under stereomicroscopy. (A) Zygote with formation ofblastodisc (arrowhead; 0.25 h post-fertilization). (B) Cleavage with some blastomeres (arrow; 1.5 h post-fertilization). (C)Blastula (arrowheads; 3 h post-fertilization). (D) Beginning of gastrula, 30% epiboly migration of blastodisc cells (arrows;6 h post-fertilization). (E) End of gastrula, blastopore closure (arrowheads; 1011 h post-fertilization). (F) Segmentation, opticalvesicle (ov), somites (arrow head) and attached tail (arrow) (16 h post-fertilization). (G) Embryo at larval stage free tail (arrow;21 h post-fertilization). (H) Recently hatched larvae (30 h post-fertilization).

composed of a continuous vestibule, with an internalcanal that narrows towards the plasmatic membraneof the egg (Ganeco & Nakaghi, 2003). According toprevious reports in teleost species, the internal canalopening allows the entrance of a single spermatozoon(Kobayashi & Yamamoto, 1981; Rizzo & Bazzoli, 1993;Ganeco & Nakaghi, 2003; Alexandre et al., 2010) as amechanism to avoid polyspermy.

In the present study, spermatozoa were firstobserved in the entrance of the micropyle immediatelyafter the mixture of sperm and oocytes even before theaddition of water (time 0), similar to the CharaciformesB. orbignyanus (Ganeco et al., 2009) and the Cyprini-formes common carp Cyprinus carpio (Kudo, 1980). Afast spermatozoa arrival (from 10 to 30 s) was reportedfor B. nattereri and Brycon spp. (Table 2). Soon after the

92 Isa et al.

penetration of the fertilizing spermatozoa to the oocyteand the membrane fusion of both gametes, membraneflow occurs at the region of sperm penetration, andprogresses toward the micropylar vestibule pluggingthe inner opening to block the supernumerary spermfrom attaching and/or to penetrating into the spacebetween egg envelope and oocyte plasma membraneforming a structure named fertilization cone (Kudo,1980; Iwamatsu et al., 1991; Brasil et al., 2002; Murata,2003). In this study, the fertilization cone was sphericaland was first observed 20 to 40 s post-fertilization,similarly to other species of genus Brycon (Table 2).The beginning of micropyle closure was observed 100 spost-fertilization, similarly to B. nattereri (Table 2). Themicropyle shape and these post-fertilization events actas mechanisms to prevent polyspermy intercepting theentry of supernumerary spermatozoa (Iwamatsu et al.,1993; Ganeco, 2003; Murata, 2003; Ganeco et al., 2009).A relatively short embryonic development period is

characteristic of neotropical rheophilic fish species, aswater temperature during spawning season is warm(Sato et al., 2003). Incubation temperature influencesthe duration of development as well as the overallsurvival rate (Sato et al. 2000; Hansen & Falk-Petersen,2002). In this study, hatching occurred 30 h post-fertilization at a water temperature of 24 C. In aprevious study using the same fish species (Andrade-Talmelli et al., 2001), hatching occurred 14 h post-fertilization at 2527 C, similar to B. cephalus and B.orbignyanus (11 to 13 h at 27 C; Table 3). The B.insignis broodfish used in both studies were selectedfrom the same Aquaculture Station at CESP, but 13years apart. Currently, the incubation period of B.insignis eggs carried out by the CESP techniciansis similar to what we have observed during ourfertilization trial. It is possible that broodfish and theirprogenies underwent domestication during all theseyears and somehow incubation period became longer.It is interesting to point out that the incubation

period of B. nattereri eggs is very long (50 to 54 h)compared with other species of this genus. However,unlike most of the neotropical fish species for whospawning occurs during the rainy season, B. nattererispawns after the rainy season up to the end of thedry season (Lima et al., 2007). Thus, water temperatureis frequently below 20 C and this delays embryonicdevelopment.The embryonic development events observed in B.

insignis are in accordance with previous observationsdescribed by Andrade-Talmelli et al. (2001) andwere similar to those reported for other species ofCharaciformes (Lopes et al., 1995; Romagosa et al.,2001; Reynalte-Tataje et al., 2004; Ninhaus-Silveiraet al., 2006; Gomes et al., 2007; Maria, 2008; Ganecoet al., 2009; Alexandre et al., 2010), as well asSiluriformes (Godinho et al., 1978; Cardoso et al.,

1995; Faustino et al., 2007; Marques et al., 2008;Amorim et al., 2009; Perini et al. 2010). During thefirst 3 h, B. insignis embryos in different stages(zygote, cleavage and blastula stage) in a givensampling time were identified. It has been reportedthat the variations in the velocity of embryonicdevelopment are related to the breeders age andthe temperature of incubation (Morrison et al., 2001).However, even within spawns fertilized and incubatedunder the same conditions, asynchrony of embryonicdevelopment has been observed in Characiformessuch as B. cephalus (Alexandre et al., 2010), B. nattereri(Maria, 2008) and P. lineatus (Ninhaus-Silveira et al.,2006) as well as in Perciformes such as the Nile tilapiaOreochromis niloticus (Morrison et al., 2001). From 3 hpost-fertilization onwards, embryos developed moreuniformly within each stage.The pattern of egg cleavage in vertebrates depends

on the amount and distribution of yolk and itsproportion in relation to the cytoplasm that com-poses the zygote (Gilbert, 1991). B. insignis eggscan be classified as macrolecithal (because of thelarge amount of yolk) or telolecithal (because yolkis concentrated at the vegetative pole) (Devlin &Nagahama, 2002). The cleavage of B. insignis followedameroblastic or partial pattern, restricted to the animalpole as commonly observed in teleosts (Lagler et al.,1977; Leme dos Santos & Azoubel, 1996; Ninhaus-Silveira et al., 2006). During the cleavage stage, thenumber of cells increased while their size decreased,as previously reported in teleost embryos (Castellaniet al., 1994; Ganeco, 2003; Ninhaus-Silveira et al.,2006; Maria, 2008; Alexandre et al., 2010).At blastula stage, the blastoderm presented a

half-moon shape and irregular spaces between theblastomeres (blastocoele) with formation of two mainregions (external cells of the blastodisc and thebeginning of yolk syncytial layer) were observed.At gastrula stage, epiboly movements began, whichextended up to the blastopore closure by theblastoderm cells and formation of the tail button. Thisevent indicates that fertilization was successful (Periniet al., 2010). The differentiation of embryonic germlayers and elongation of the embryo at the headtailaxis were developed at segmentation stage. The releaseof the tail and the formation of more than 26 somitescharacterized the beginning at larval stage. During thisstage, muscle contractions were observed. At hatching,muscle contractions are intensifies which culminateswith chorion rupture. The blastula, gastrula, segment-ation, larval and hatching stages were similar to thedescription of the embryonic development not only ofother Characiformes (Castellani et al., 1994; Andrade-Talmelli et al., 2001; Ganeco, 2003; Ninhaus-Silveiraet al., 2006; Maria, 2008; Alexandre et al., 2010), but alsoof Siluriformes (Marques et al., 2008; Perini et al., 2010)

Oocytes and embryos in Brycon insignis 93

and Cypriniformes (Kimmel & Law, 1985; Trinkaus,1984).The morphology of oocyte, post-fertilization events

and embryonic development stages here observedare in accordance with similar studies previouslycarried out in B. insignis, in other species ofCharaciformes, as well as Cypriniformes, Perciformesand Siluriformes. Such knowledge is important fora better understanding of reproductive features ofa species, helpful to elucidate issues related to fishrearing at this stage, useful to further taxonomic,ecological and conservational studies in the B. insignis.

Acknowledgements

This research was supported by FAPEMIG (CVZ160906; APQ 257850407; CAEG APQ 0271502),CNPq (556495/20080; 141748/20087) and ANEELP&D CESP (0061017/2006), and is part of the firstauthors PhD project. The authors warmly thank D.Caneppele and L.H.C. Oliveira of the Hydrobiologyand Aquaculture Station of CESP, Paraibuna/SP forassistance with experiments.

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