GAMETOGENESIS AND REPRODUCTION OF THE MATRINXÃ Brycon orthotaenia ( GÜNTHER , 1864 ) ( PISCES : CHARACIDAE ) IN THE SÃO FRANCISCO RIVER , MINAS GERAIS , BRAZIL GONÇALVES

The gametogenesis and reproduction of the matrinxã, Brycon orthotaenia in the São Francisco river, Pirapora Region, Minas Gerais, were studied from December, 1998 to November, 1999. The matrinxã B. orthotaenia is an endemic species in the São Francisco river basin in Brazil and important for commercial fishing. The size of the 1 gonadal maturation, indicated by the total length of the smallest spent males and spawned female, was 32.0 and 40.5 cm respectively. Reproduction occurred from October to January, when high frequencies of females and males at an advanced ripening/mature stage and spawned females were found. The short spawning period and histological characteristics of spawned ovaries containing only oocytes in stages of initial development, along with post-ovulatory and atresic follicles, indicated that Brycon orthotaenia presents total spawning.


INTRODUCTION
Human population growth and an increase in agricultural and industrial activities, as well as dam construction, mineral extraction, deforestation and predatory fishing cause changes in the environment that directly affect fish.As a result of this environment degradation, the São Francisco river, which was one of the main fishing resources in Brazil, is presently a fishery in decline (Godinho & Godinho, 2003).Taking this into consideration, the matrinxã, Brycon orthotaenia (Günther, 1864) known previously as B. lundii Reinhardt, 1874, an endemic species in the São Francisco river basin and important for commercial fishing, reaching 7 kg of body weight, is threatened with extinction in some regions of the São Francisco basin (Sato, 1999).
The knowledge about different stages of fish gonadal maturation provides important information necessary to prohibit fishing during the reproduction period; allowing the fishery stock to recover (Noble & Jones, 1993).Establishing the reproduction period and length of gonadal maturation, using microscopic criterion justify implementing fishery legislation more accurately (Bazzoli, 2003).
The present work outlines the stages and size of 1 st gonadal maturation, the reproductive period and type of spawning of the matrinxã, B.orthotaenia in the São Francisco river, Pirapora Region, Minas Gerais, Brazil.

The fish
A total of 75 specimens, 55 females and 20 males of the matrinxã B. orthotaenia were collected by fishermen using casting and drifting nets between December, 1998 andNovember, 1999 in the São Francisco river, Pirapora Region (17° 20' 45'' S and44° 56' 55'' W), state of Minas Gerais, Brazil.Each fish was measured (total and standard length), weighed (body weight) and sexed.

Light microscopy
Fragments of gonads from all specimens were fixed in Bouin's fluid for 4-12 h and subjected to routine histological techniques: embedded in paraffin, sectioned at 3 a 5 m and stained with hematoxylin/eosin (HE), in the Ichthyology Laboratory, Graduate Program in Zoology of Vertebrates, Pontifical Catholic University of Minas Gerais.

Stages of gonadal maturation and type of spawning
Microscopically, the stages of gonadal maturation were determined based on the distribution of oocytes and spermatogenic cells.The type of spawning was identified based on histological characteristics of spawned ovaries as well as frequency distribution of the stages of gonadal maturation (Bazzoli, 2003).

Size of 1 st gonadal maturation
The length of the 1 st gonadal maturation was indicated by the total length of the smallest regression male and female.

Morphology of ovaries and oogenesis
The ovaries of B. orthotaenia were paired, elongated organs located in the coelomic cavity laterally to the digestive tube, ventrally to the kidneys and ventro-laterally to the gas bladder.The ovaries were attached to the gas bladder through the mesovarium and joined at their caudal ends to form the common ovarian duct, which opened at the urogenital papilla.Macroscopically, the ovaries presented variations in the volume, thickness and color, according to the stages of gonadal maturation.Microscopically, they were wrapped by the tunica albuginea that emits septae to the inside of the organ, forming ovigerous lamellae in which there were oocytes in different development stages.

Morphology of testes and spermatogenesis
The testes of B. orthotaenia were paired, elongated and fusiform organs located in the coelomic cavity laterally to the digestive tube, ventrally to the kidneys and ventro-laterally to the gas bladder.The testes were attached to the gas bladder through the mesorquium and joined at their caudal ends to form the common spermatic duct, which opened at the urogenital papilla, where the spermatozoa leave the body.Microscopically, the testes were wrapped by the tunica albuginea that emits septae to the inside of the organ, forming lobules filled with semniferous tubules.The wall of these tubules was made up of cysts that were delimited by cytoplasmatic elongations of Sertoli cells.In each cyst, spermatogenic cells in the same development stage were found.
During spermatogenesis, alterations in primordial germ cells or spermatogonias give origin to spermatozoa.Based on the histological characteristics, six types of cells were identified during the spermatogenic development: primary spermatogonia, secondary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa (Fig. 11)

TABLE 1 Macro and microscopic characteristics of stages of gonadal maturation (SGM) of females and males of Brycon orthotaenia
in the São Francisco river, state of Minas Gerais between December, 1998 and November, 1999.

SGM Morphologic characteristics Ovaries
Testes 1 • Translucent and not very voluminous; • Presence of O1 and O2.
• Thin and translucent; • Lumen of seminiferous tubules closed and wall made up of spermatogonias.2 • Greenish, voluminous and some oocytes visible to the naked eye; • Presence of O1, O2, O3.
• Whitish and voluminous; • Lumen of seminiferous tubules open with small quantity of spermatozoa and wall made up of cysts of all spermatogenic cells.3 • Dark greenish to purple, maximum volume and numerous oocytes visible to the naked eye; • Presence of O1, O2, O3 and mainly O4.
• Milky-whitish and maximum volume; • Lumen seminiferous tubules with large quantity of spermatozoa and acidophyle secretion.4 • Flaccid and hemorrhagic, with some oocytes visible to the naked eye; • Presence of O1, O2, post-ovulatory follicles, in addition to rare O3 and O4 in follicular atresia.

Frequency of stages of gonadal maturation and type of spawning
High frequencies of females in the advanced maturation/mature and regression stages were recorded during October/November and December/ January.Males in the advanced maturation/mature stage occurred only during October/November and December/January, while ones in the regression stage were not captured only during April/May and June/July (Fig. 17).
The short spawning period and histological characteristics of the regression ovaries, containing only initial and advanced perinucleolar oocytes, as well as post-ovulatory and atresic follicles, indicated that B. orthotaenia presents total spawning.

Length of 1 st gonadal maturation
The smallest male and female captured in the advanced maturation/mature stage measured 30.0 and 33.5 cm of total length, respectively.The smallest regression male and female measured 32.0 and 40.5 cm of total length, respectively (Tables 2  and 3).

DISCUSSION
In this work, the small number of captured specimens may be related to the fact that B. orthotaenia is probably among the endangered species of the São Francisco river (Lins et al., 1997), especially in some regions, such as downstream of the Três Marias storage dam, and upstream of the Sobradinho storage dam to the Atlantic Ocean (Sato, 1999).
The macroscopic morphology of the gonads of B. orthotaenia was similar to reports from Zaniboni-Filho (1988) and Romagosa et al. (1999) for Brycon cephalus, an abundant and economically important fish found in the Amazon river basin in Brazil.The morphological changes observed in the nucleus, cytoplasm and oocyte envoltories of B. orthotaenia during oogenesis, especially in relation to the yolk nucleus, cortical alveoli, zona pellucida and follicular cells, coincide with the findings of Bazzoli (1992) in other fish of the genus Brycon.After spawning, post-ovulatory follicles were formed, which in B. orthotaenia, were made up of hypertrophied follicular cells and thin theca, similar to Astyanax bimaculatus lacustris described by Drummond et al. (2000).These authors have also observed programmed cell death or apoptosis during the involution of the follicles.According to Guraya (1986) and as recorded in this study, even though atresic follicles may be formed at any stage of the reproductive cycle and are more frequent  in regression ovaries, non-ovulated vitellogenic oocytes have begun the process of absorption or atresia.The morphological event occurred when atresic follicles of B. orthotaenia were being formed and are compatible with the reports from Miranda et al. (1999).
The wall of the seminiferous tubules of B. orthotaenia was made up of cysts of germ cells at different stages of spermatogenesis and in each cyst, delimited by cytoplasmatic elongations of Sertoli cells, germ cells were at the same development stage, a common pattern in most teleosts (Grier, 1981;Lo Nostro et al., 2003).The basic organization of the testes and morphologic characteristics of spermatogenic cells are common to all fish as well as other vertebrates (Billard et al., 1982).The origin of teleost spermatogenic cells is controversial.In the present study, there were indications of the origin of these cells from spermatogonias, due to their presence in the testes in the whole reproductive cycle, which is compatible with Grier (1981).Spermatogenesis of B. orthotaenia occurred, inside the cysts, synchronously with the release of spermatozoa into the lumen of seminiferous tubules, a common pattern in fish of the genus Brycon (Zanoboni-Filho & Resende, 1988;Romagosa et al., 1999;Nogueira, 2000).However, in some Characids, the cysts hatch when in the spermatid stage (Andrade et al., 2001) Nogueira (2000) using classic histological techniques detected neutral glicoproteins in the acidophyle secretion of the seminiferous tubules of B. orthotaenia.This type of secretion was also reported for other Brazilian Characiforms (Bazzoli, 2003), and due to its histological nature it may act as an energy resource to spermatozoa, similar to seminal vesicles found in some Siluriformes groups (Santos et al., 2001).
In this study, four stages of the reproductive cycle were determined, however, in the literature, the number of these stages varied from three to eleven.This disagreement may be due to the methodology used instead of morpho-functional differences of the gametogenesis process, which seems to have a similar development among teleosts (Bazzoli, 2003).The reproduction period of fish in the São Francisco river around the city of Pirapora begins in October (Aguirre, 1936).In fact, in this work the peaks of females and males in advanced maturation/ mature and regression females occurred between October and January.Brito & Bazzoli (2003) studied the reproduction of the surubim, Pseudoplatystoma coruscans, in the same area and confirmed that the reproduction peak also occurs between October and January, when water turbidity (mean 300 ntu) and pluviometry (mean 100 mm) were high.
The present study indicated that the spawning of Brycon orthotaenia was of the total type, which seems to be characteristic among fish of the genus Brycon (Zaniboni-Filho & Resende, 1988;Romagosa et al., 1999).
With respect to the size of 1 st gonadal maturation of B. orthotaenia in the São Francisco river, Pirapora region, state of Minas Gerais, it is important to report the impossibility of using the L-50 method, that establishes the length in which 50% of the population is adult and 50% is immature (Vazzoler, 1996), due to the legislation that regulates fishing activity by not allowing the capturing of immature fish.Therefore, in this work the length of gonadal maturation was suggested based on the total length of the smallest regression male and female, similar to the proceedings used by Bazzoli (2003).