Introduction
The ‘curimbata’, Prochilodus lineatus, a member of the family Prochilodontidae, is an iliophagous fish species with wide distribution over southeastern Brazil that undergoes reproductive migration and total spawning (Fowler, Reference Fowler1951). According to Corrêa & Castro (Reference Corrêa e Castro1990), this species is recorded along the entire Paraná–Paraguay and Paraiba river basins. It represents an economically and ecologically important native species of medium to large size.
Knowledge of the embryonic development of different fish species is useful to the management of fishery resources, as well as to surveys related to fish culture, since it provides additional information about species' life cycles. In addition, embryological analysis is helpful in studies about evolutionary relationships, heredity, developmental mechanisms and environmental influences over structural features of distinct organisms (Lagler, Reference Lagler1959).
The embryonic development of fishes is a complex phenomenon, useful for ontogeny studies, experimental modelling, and evaluation of environmental quality and effects of toxic substances on aquatic fauna (Flores et al., Reference Flores, Araiza and Valle2002), as well as for experiments on ex situ species preservation.
Information about the embryology of the curimbata is scarce and restricted to reports such as those by Brazil et al. (Reference Brasil, Nakaghi, Leme dos Santos, Quagio-Grassiotto and Foresti2002), which studied egg morphology modifications just after fertilization, and that of Castellani et al. (Reference Castellani, Tse, Leme dos Santos and Faria1994), who carried out observations of the embryonic development of P. lineatus under the light microscope.
Therefore, given on the ecological and economic value of Prochilodus lineatus, the present work was performed to analyse the morphological events during the embryonic development of this species at the structural and ultrastructural levels.
Materials and methods
Adult individuals of Prochilodus lineatus from the broodstock at the Aquaculture Division of the Faculdade de Medicina Veterinária e Zootecnia, UNESP, Botucatu, São Paulo, Brazil were used to obtain embryos.
Prochilodus lineatus, like many other Brazilian freshwater species, requires an upstream migration to spawn (Godoy, Reference Godoy1975). In captivity, this species is not able to reproduce naturally and hormonal induction is necessary. Breeders were stimulated with carp pituitary extract by inoculating 3 mature females (3 years old) with 0.5 mg and 5 mg/kg body weight, respectively, at an interval of 10 h, and 6 mature males (2 years old) with 1 mg/kg body weight at the time of the second female inoculation. The extrusion of eggs and sperm was performed about 6 h after the last induction.
The dry method was employed for the fertilization process, in which the eggs are mixed with the sperm avoiding contact with water. After that, water was added in order to activate spermatozoa and allow egg hydration. The excess semen was rinsed off and the eggs were incubated in 200 l vertical incubators. To verify a putative effect of water temperature on embryo development, the eggs were divided into two groups incubated at different temperatures (24 °C and 28 °C). The incubators were connected to a closed heated water system coupled with a thermostat.
Embryonic development
To evaluate the possible temporal morphological variation of P. lineatus embryos, about 200 embryos were collected at different development stages, defining the moment of fertilization as time zero. The first samples were collected within intervals of 15 and 10 min for eggs incubated at 24 and 28 °C, respectively, until 2 h of embryonic development, while subsequent samples were taken at intervals of 1 h until the point of hatching. The embryo samples were fixed in a solution of 2% glutaraldehyde, 2% paraformaldehyde diluted in sodium phosphate buffer 0.1 M, pH 7.3, for 24 h prior to analyses.
Fifty prefixed embryos were selected for in toto analysis. The chorion was extracted using watchmaker's forceps and a needle, and then the embryos were stained with Harries haematoxylin–eosin (H&E) prior to being analysed and photographed using a stereomicroscope.
Stuctural and ultrastructural analysis
Twenty representative individuals from each development stage were carefully selected and embedded in glycol methacrylate. These samples were submitted to microtomy to obtain serial transverse and longitudinal cuts of from 3 to 5 µm. After that, they were stained with Harries haematoxylin–eosin or toluidine blue and analysed and photographed using a Zeiss Axiophot photomicroscope.
The embryos were postfixed in 1% osmium tetroxide for 2 h, counterstained with an aqueous solution of 0.5% uranyl acetate, dehydrated with acetone and embedded in epoxy resin for analysis by transmission electron microscopy (TEM). The ultrafine sections were caught on a copper net, counterstained with uranyl acetate (Watson, Reference Watson1958), washed in 50% alcohol and re-counterstained in lead citrate (Reynolds, Reference Reynolds1963). The material was analysed and electromicrographed using a Philips CM100 transmission electron microscope.
For analyses by scanning electron microscopy (SEM), the embryos, prefixed in 2.5% glutaraldehyde, were transferred to a 13 mm coverslip, embedded with 1% poly-L-lysine, postfixed in 0.5% osmium tetroxide, dehydrated with ethanol and dried in a critical-point dryer (Balzers CPD-20). The samples were covered with a 10 mm gold pellicle in a Balzers Metalizer MED-010 and observed and electromicrographed using a Philips 515 scanning electron microscope.
Results
Embryogenesis
The duration of embryonic development in Prochilodus lineatus, from fertilization until hatching, has been shown to be dependent on the water temperature. At 24 °C, the incubation period was 22 h, and at 28 °C, it was 14 h. The following stages were identified in the embryonic development of P. lineatus after fertilization: zygote, cleavage, blastula, gastrula, segmentation, larval and hatching (Tables 1, 2; Figs. 1, 2, 3).
Embryonic development of Prochilodus lineatus at 24 °C
Embryonic development of Prochilodus lineatus at 28 °C
Phases of the embryonic development of Prochilodus lineatus. (A), (A') Post-fertilization without chorion; (B), (B') 2-cell embryo; (C), (C') 4-cell embryo; (D), (D') 8-cell embryo; (E), (E') 16-cell embryo; (F), (F') 32-cell embryo; (G), (G') 64-cell embryo; (H) morula; (I) gastrula (25% epiboly); (J) gastrula (50% epiboly); (K) gastrula (75% epiboly); (L), (L') gastrula (90%). Scale bars represent 113.6 μm.
(A), (A') Neurula; (B) in the presence of about 13 somites, optic vesicle, attached tail; (C) embryo bearing nearly 19 somites, optic vesicle, Kupffer's vesicle and attached tail; (D) 24 somites, presence of optic and otic vesicles, absence of Kupffer's vesicle and free tail. s, somite; arrow, neural keel; vk, Kupffer's vesicle; op, optic vesicle. Scale bars represent: (A), (A') 55 μm; (B) 57.1 μm; (C) 70.4 μm; (D) 76.3 μm.
(A) 30+ somites, growing larva; (B) pre-hatchery embryo; (C) hatched embryo. Scale bars represent: (A) 63.3 μm; (B) 75.6 μm; (C) 69.4 μm.
A higher heterogeneity in embryo development was observed at 24 °C, i.e. embryos at different stages of embryogenesis were detected at the same time, especially at the beginning of cleavage (1–1.5 h) (Fig. 4A). Furthermore, embryos displaying 5, 7, 10, 12 or 15 blastomeres were also found (Table 1; Fig. 4A, B). At 28 °C, embryo development was homogeneous, despite slight variations during the first cleavage phases (0.66–1.66 h) (Table 2; Fig. 4C, D).
Analysis of the embryonic development of Prochilodus lineatus under two temperature conditions (24 °C and 28 °C). (A), (B) Segmentation period. (C), (D) Morphogenesis period. s/s, not segmented; bl, blastomeres; epi, epiboly.
1: Zygote stage (0–0.75 h (24 °C); 0–0.34 h (28 °C))
After fertilization, hydration of the eggs could be observed by the increase in the perivitelline space, pronuclear fusion, and cytoplasm reorganization with the establishment of vegetal and animal poles (Fig. 5A–C). The animal pole was composed of active cytoplasm and a nucleus, allowing in vivo and light microscopic identification, since it is slightly transparent. On the other hand, the vegetal pole was denser at in vivo observation and weakly stained in total preparations (Fig. 5C), being composed of yolk vesicles (Fig. 6A–C). Moreover, a thin layer of cytoplasm involving the whole yolk was observed, comprising several central alveoli reminiscent of the cortical reaction during fertilization (Fig. 6A).
(A) Fertilized and non-hydrated egg (×129); (B), (C) hydrated egg, showing well-defined animal and vegetal poles (×55). arrowhead, chorion; *, perivitelline space; arrow, animal pole; v, yolk. Scale bars represent: (A) 77.5 μm; (B), (C) 181.8 μm.
Analysis under a light microscope of embryos of Prochilodus lineatus, stained with basic toluidine blue. (A) 0.25 h after fertilization, showing the yolk cytoplasmic layer; (B) cleavage phase (1.5 h), revealing the penetration of yolk globules into blastomeres; (C) detail of the formation of the yolk syncytial layer in an embryo at the blastula stage; (D) 4.25 h of development (gastrula stage), characterized by the presence of blastomeres with euchromatic nuclei, yolk syncytial layer and high mitotic activity (MO); (E) embryo section at 50% of epiboly, stained with basic toluidine blue; (F) embryo section at 90% of epiboly, stained with H&E. gv, yolk globules; ycl, yolk cytoplasmic layer; b, blastomere; bl, blastoderm; ysl, yolk syncytial layer; n, nucleus; gv, yolk globules. Scale bars represent: (A) 11.5 μm; (B) 17.5 μm; (C) 16.7 μm; (D) 3.6 μm; (E) 80 μm; (F) 5.7 μm.
2: Cleavage stage (1.0–2.25 h (24 °C); 0.50–1.34 h (28 °C))
The cleavage stage was characterized by the beginning of mitotic division, and it continued until the formation of irregular spaces among internal cells, which might be considered a kind of blastocoele. In P. lineatus, the cleavages are meroblastic and can be described as follows: the first cleavage plane was vertical, giving rise to 2 blastomeres; the second plane was vertical and perpendicular to the first, giving rise to 4 blastomeres; the third was vertical and parallel to the first, giving rise to 8 blastomeres displaying a 4 × 2 arrangement; the fourth was vertical and parallel to the second, originating 16 blastomeres in a 4 × 4 formation; the fifth plane was vertical and parallel to the first cleavage, originating 32 blastomeres in a 4 × 8 formation; and the sixth cleavage plane was horizontal, giving rise to two cell layers comprising 64 blastomeres (Fig. 8A–H).
For as long as the cleavages occur, the number of blastomeres increases and their size decreases. Until the third cleavage the cells keep their homogeneity and, after the fourth cleavage, blastomeres of distinct sizes can be observed (Fig. 8A–H).
No distinctive layer was observed between the blastoderm and yolk. It was verified that yolk globules penetrate into blastomeres in a fragmented way, probably to facilitate their absorption by cells (Fig. 7C). Analyses under the light microscope showed that, during the cleavage stage, individualized nuclei were absent (Fig. 6C) while TEM showed blastomeres with a large number of mitochondria, euchromatic nuclei and free ribosomes (Fig. 7B, C), indicating a high cell metabolism, typical of high mitotic activity.
Analysis under an electron microscope of embryos of Prochilodus lineatus. (A) Embryo blastomeres at cleavage stage, showing euchromatic nucleus and a large number of yolk vesicles in the cytoplasm (TEM); (B) Detail of yolk globules (SEM); (C) Embryos of P. lineatus at the gastrula stage showing the periblast with euchromatic nucleus, cytoplasm with several vesicles, mitochondria and some yolk granules (TEM); (D) ultrastructure (TEM) showing irregular nuclei of the yolk syncytial layer and subjacent yolk globules. n, euchromatic nucleus; gv, yolk globules; b, blastomeres; ysl, yolk syncytial layer. Scale bars represent: (A) 6.1 μm; (B) 20.4 μm; (C), 3.8 μm; (D) 5.7 μm.
Observation of the first six cleavage planes in embryos of Prochilodus lineatus under a scanning electronic microscope. Scale bars represent 263.1 μm.
3: Blastula stage (3.0–4.0 h (24 °C); 1.66–2.0 h (28 °C))
At the beginning of the blastula stage, a dome-shaped blastoderm was present. The cells continuously underwent divisions, but the cleavage planes were undetermined. As the number of cell increased, the blastoderm changed into a half-moon shape.
The main characteristics of this stage are the irregular spaces among blastomeres (blastocoele) and the beginning of the formation of a periblast or yolk syncytial layer (Fig. 7D, E). At the end of this stage, the first epiboly movements could be identified.
4: Gastrula stage (4.0–8.0 h (24 °C); 3.0–6.0 h (28 °C))
The gastrula stage was characterized by epiboly movement and the occurrence of morphogenetic movements of convergence and cell migration that give rise to the first layers and to the head–tail and latero-lateral embryonic axes.
The epiboly movement started after 4 h at 24 °C and after 3 h at 28 °C. It was observed as a fringe, formed by the yolk syncytial layer, across the blastoderm border, from its formation to the closure of the blastopore (Fig. 6C, E, F). The morphogenetic movements of convergence and cell migration began at the border of blastoderm, at about 50% of epiboly (Fig. 6E), originating the germ ring and the embryonic shield and culminating with the formation of the two embryonic layers, the epiblast and hypoblast.
The epiboly movement goes on alongside the closure of the yolk plug by the yolk syncytial layer, which is delimited by the blastopore (Fig. 6F), following the total recovery of the blastoderm plug.
Ultrastructural analyses demonstrated that the blastomere cytoplasm contains a large number of mitochondria and vesicles, several filled with yolk material. These yolk granules, prior to absorption by blastoderm cells, are fragmented at the periblast region. The blastomeres and periblast nuclei were euchromatic (uncondensed), indicating high metabolic activity (Fig. 7C, D).
5: Segmentation and organogenesis stage (10.0–15.0 h (24 °C); 7.0–10.0 h (28 °C))
The stage of segmentation and organogenesis was characterized by the formation of rudimentary organs and systems from the embryonic layers. Thus, the somites, the notochord and the neural tube, as well as the initial delimitation of the intestines, were observed, leading to the subsequent growth and elongation of the embryo, particularly along the head–tail axis.
After 10 h of development at 24 °C and 7 h at 28 °C, it was possible to identify the neural keel, the neural plate, the mesendoderm notochord (Fig. 9A) and Kupffer's vesicle at the tail region (Fig. 9B), as well as segmented somites in some embryos (28 °C) (Fig. 2A).
Details under a light microscope of embryos of Prochilodus lineatus at the segmentation stage. (A) Section showing the notochord, the neural keel, the mesendoderm and the neural plate (H&E); (B) detail of the structure of Kupffer's vesicle; (C) longitudinal section of the optic vesicle (H&E); (D) longitudinal section of somites (toluidine blue); (E) transverse section, detailing the notochord, somites and the neural tube (H&E); (F) longitudinal section detailing the presence of the optic vesicle (H&E). gv, yolk globules; ysl, yolk syncytial layer; no, notochord; ot, optic vesicle; tn, neural tube; s, somite; me, mesendoderm; pn, neural plate; sn, neural keel; vk, Kupffer's vesicle. Scale bars represent: (A) 7.5 μm; (B) 7.7 μm; (C) 9.1 μm; (D) 14.6 μm; (E) 22.5 μm; (F) 23.4 μm.
In this phase, the formation of neural tube is initiated (Fig. 10E) and as long as its components show differential growth, it is possible to identify the prosencephalon, mesencephalon and rhombencephalon regions (Figs. 2B, 9A).
Longitudinal sections of embryos of P. lineatus at the larval stage, stained with H&E. (A) Detail of the optic calyx and the crystalline lens; (B) somites at myogenesis; (C) details of somites and notochord; (D) details of the optic vesicle and the regions comprising the prosencephalon, mesencephalon and rhombencephalon; (E) general details of the embryo, revealing a primitive gut. gv, yolk globules; ysl, yolk syncytial layer; no, notochord; co, optic calyx; c, crystalline lens; s, somite; ot, optic vesicle; mes, mesencephalon; rom, rhombencephalon; pro, prosencephalon; ip, rudimentary intestine. Scale bars represent: (A) 18.2 μm; (B) 39.1 μm; (C) 18.1 μm; (D) 51.5 μm; (E) 130.2 μm.
After 12 h of development at 24 °C and 8 h at 28 °C, the embryos already contained an optic vesicle and several somites (Fig. 9D, F). After 15 h at 28 °C and 10 h at 24 °C, the otic vesicle was present, as well as a complete neural tube (Fig. 9C, D), a rudimentary digestive system and free tail; in addition Kupffer's vesicle was absent (Fig. 2D).
6: Larval stage (16.0–21.0 h (24 °C); 11.0–13.0 h (28 °C))
A free tail, the presence of more than 25 pairs of somites and a ready-to-hatch larval shape, characterized the embryos at the larval stage. The embryos showed a well-developed optic calyx, crystalline lens and optic vesicle (Fig. 10A, D). The notochord extended from the cephalic region to the tail (Fig. 10E). The somites showed the beginning of the myogenesis process for the formation of muscles (Fig. 10B, C) and the posterior primitive intestine was well defined (Fig. 10E). Another feature of this stage is the occurrence of spasmodic movements, which increased as embryonic development proceeded.
Discussion
The eggs produced by Prochilodus lineatus are pelagic (Nakatani et al., 2001), non-adhesive, greenish, slightly transparent, and show a large perivitelline space after hydration. According to their quantity of yolk and its location, they can be classified as macrolecithal (containing a large amount of yolk) or telolecithal (yolk concentrated at the vegetal pole), while the cytoplasm and its organelles are concentrated at the animal pole. These features are similar to those described for species of the genus Brycon (Ganeco, Reference Ganeco2003; Andrade-Talmelli et al., Reference Andrade-Talmelli, Kavamoto, Romagosa and Fenerich-Verani2001; Romagosa et al., Reference Romagosa, Narahara and Fenerich-Verani2001; Eckmann, Reference Eckmann1984).
Fat droplets inside the yolk sac were absent in P. lineatus, similar to what has been observed in other Characiformes, such as Brycon orbignyanus (Ganeco, Reference Ganeco2003) and Brycon insignis (Andrade-Talmelli et al., Reference Andrade-Talmelli, Kavamoto, Romagosa and Fenerich-Verani2001).
The morphological events identified during the embryogenesis of P. lineatus, as well as the short duration of embryonic development, were similar to those reported in other teleosts (Ganeco, Reference Ganeco2003; Flores et al., Reference Flores, Araiza and Valle2002; Andrade-Talmelli et al., Reference Andrade-Talmelli, Kavamoto, Romagosa and Fenerich-Verani2001; Cardoso et al., Reference Cardoso, Alves, Ferreira and Godinho1995; Kimmel et al., Reference Kimmel, Ballard, Kimmel and Ullmann1995; Ribeiro et al., Reference Ribeiro, Leme dos Santos and Bolzan1995).
The pattern of egg segmentation in vertebrates depends on the amount and distribution of yolk and its proportion in relation to the cytoplasm that composes the blastodisc (Gilbert, Reference Gilbert1991). The cleavage of P. lineatus follows a meroblastic or partial pattern, as commonly observed in most teleosts (Lagler et al., Reference Lagler, Bardach, Miller and Passino1977, Leme dos Santos & Azoubel, Reference Leme dos Santos and Azoubel1996). The arrangement of blastomeres (4 × 2; 4 × 4; 4 × 8) at the early cleavage stages is similar to that reported in Catostomus commersoni (Long & Ballard, Reference Long and Ballard1976), Danio rerio (Kimmel et al., Reference Kimmel, Ballard, Kimmel and Ullmann1995) and Oreochromis niloticus (Morrison et al., Reference Morrison, Miyake and Wright2001), but it differs from those observed in Rhamdia sapo (Matkovik et al., Reference Matkovic, Cussac, Cukier, Guerrero and Maggese1985), Alosa sapidissima (Shardo, Reference Shardo1995) and Brycon orbignyanus (Ganeco, Reference Ganeco2003).
During the cleavage of fish embryos the number of cells increases while their size decreases, a feature previously reported by Castellane et al. Reference Castellani, Tse, Leme dos Santos and Faria(1994) in P. lineatus and also observed in the present study.
Morrison et al. (Reference Morrison, Miyake and Wright2001) suggest that the variations in the rate of embryogenesis and embryo development (asynchrony and malformations) are related to the breeders' age and the temperature of incubation. Kimmel et al. (Reference Kimmel, Ballard, Kimmel and Ullmann1995) and Morrison et al. (Reference Morrison, Miyake and Wright2001) stated that, even within spawns fertilized and incubated under optimal conditions, there is asynchrony of embryonic development. Hisaoka & Firlit (Reference Hisaoka and Firlit1960) reported that, in zebrafish, the mitotic divisions are synchronous until 32 cells but become asynchronous at 64 cells. Arezon et al. (Reference Arezon, Lemos and Bohrer2002) reported that embryos of Cynolebias melanotaenia develop faster at 25 °C, but severe abnormalities occur, in contrast to what is detected at lower temperatures (20 and 16 °C).
In the present experiment with P. lineatus, despite the utilization of young breeders (2 years old) and a constant water temperature, incubation at 24 °C led to a higher asynchrony of embryonic development and a variation in blastomere division, in some cases, following a non-geometric progression after the third and fourth planes. At 28 °C this variability was reduced and nearly restricted to the cleavage stage. In addition, cleavage progression was geometric, at least until the first 64 cells. These data corroborate the observations of previous reports and stress the important role of incubation temperature in the embryonic development of fish species.
Hisaoka & Firlit (Reference Hisaoka and Firlit1960) suggested that the protoplasmic movement between the yolk and blastomeres ceases after the 64-cell stage. This fact could be related to the beginning of periblast formation, which occurs at the beginning of the blastula stage and morula stage in P. lineatus.
In this species the formation of a blastocoele has also been observed, visible as irregular spaces among some blastoderm cells, as described by several authors (Kimmel & Law, Reference Kimmel and Law1985; Trinkaus, Reference Trinkaus1984; Kimmel et al., Reference Kimmel, Ballard, Kimmel and Ullmann1995; Ganeco, Reference Ganeco2003) in other teleost species. However, in some species, such as Oreochromis niloticus (Morrison, Reference Morrison, Miyake and Wright2001), no characteristic cavity was identified, whereas others, such as Hippocampus reidi (Silveira, Reference Silveira, Garcia and Fernandes2001) and trout (Lagler, Reference Lagler, Bardach, Miller and Passino1977), a typical blastocoele cavity was seen between the blastoderm and the periblast.
The gastrula stage begins with the first epiboly movements (Leme dos Santos & Azoubel, Reference Leme dos Santos and Azoubel1996) and is completed by closure of the blastopore by the blastoderm and the formation of a tail button (Kimmel et al., Reference Kimmel, Ballard, Kimmel and Ullmann1995). These observations are in accordance with what was detected in P. lineatus and also reported by Ganeco (Reference Ganeco2003) in Brycon orbignyanus. On the other hand, in Oreochromis niloticus, Morrison et al. (Reference Morrison, Miyake and Wright2001) reported that, due to the size of yolk, the embryo is not able to extend over the entire vegetal pole and, thus, rudimentary organogenesis (somite segmentation) starts before the epiboly movement is finished.
In Prochilodus lineatus, the formation of the embryonic shield is visible when the blastoderm involves 50% of the yolk sphere, similar to previous observations in other species (Kimmel, Reference Kimmel, Ballard, Kimmel and Ullmann1995; Firlit & Hisaoka, Reference Hisaoka and Firlit1960; Ganeco, Reference Ganeco2003).
According to Hisaoka & Firlit (Reference Hisaoka and Firlit1960), Kupffer's vesicle represents a remnant structure of the archenteron that is located over the periblast and below the notochord. In Oncorhynchus keta (Mahon & Hoar, 1956 cited in Hisaoka & Firlit, Reference Hisaoka and Firlit1960) Kupffer's vesicle is described as an oblique and elongated cavity, with walls of columnar epithelium, which is separated from the periblast by a layer of endoderm cells. The histology of this structure in P. lineatus was similar to that reported in O. keta.
Following Kimmel (Reference Kimmel, Ballard, Kimmel and Ullmann1995), the neurula phase was included in the segmentation stage. Brummett & Dumont (Reference Brummett and Dumont1978) and Morrison (Reference Morrison, Miyake and Wright2001) identified Kupffer's vesicle in the early phases of the segmentation stage. In the present work, this vesicle was observed at the phase of 13 somites and disappeared after the 24-somite stage; its function remains unknown. However, Brummet & Dumont (Reference Brummett and Dumont1978) hypothesized that it could have a digestive function, favorable to yolk resorption, since several ciliated cells were observed inside Kupffer's vesicle and the intestines of Fundulus heteroclitus.
According to Silveira (Reference Silveira, Garcia and Fernandes2001), the ectoderm on the notochord is transformed into a neural plate, which becomes centrally depressed giving rise to the neural keel, which following its posterior enclosure by the fusion of neural filaments, originates the neural tube. As observed in P. lineatus, the prosencephalon, mesencephalon and rhombencephalon regions developed from the posterior region of the neural tube, corroborating the description of the embryonic development of Brycon orbignyanus carried out by Ganeco (Reference Ganeco2003).
Acknowledgements
This work was supported by Aquaculture Division of the Faculdade de Medicina Veterinária e Zootecnia, UNESP, Botucatu, São Paulo, Brazil, which provided the fish and the facilities used in this study, and also by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Pesquisa).
