Reproductive biology and variations in the gonadal development of the fish Curimatã ( Prochilodus brevis Steindachner , 1875 ) in captivity

This study analyzed the reproductive biology of Prochilodus brevis held captivity during ontogenetic development in order to contribute to the conservation and improvements to induced breeding of this specie, which stands out for having commercial value attributed to the consumption of his "ova" and to be endemic in the Northeast. Sampling was fortnightly and 137 specimens were analyzed. Observations macroscopic and microscopic of the gonads allowed the identification of four stages of gonadal maturation: Immature, In Maturation, Mature and Regression. The age at maturity was predicted with a satisfactory degree of confidence, because the gonadal development was observed from fingerlings until adulthood. Hatching of fingerlings was accompanied at Estevão de Oliveira Fishery Station, in Caicó/RN/Brazil. Later the fingerlings were transferred to a tank in the Agricultural School of Jundiaí in Macaíba/RN/Brazil. Were verified juvenile males and females up to 225 days post-hatching (dph), in maturing stage from 247 dph, individuals with mature gonads from 274 dph and in regression stage from 359 dph for females and 410 dph for males, being corroborated by the variation in the curve of maturation based on the values of the Gonadosomatic Index. Thus, it was observed that even in captivity P. brevis reaches all stages of gonadal maturation, except for spawning, due to the absence of appropriate environmental conditions, since it is a species rheophilic. Therefore, it was found that the maturation of P. brevis occurs between 247 and 340 dph, corresponding to the period from October to January, similar to the natural environment.


Introduction
Fish belonging to the order Characiformes are currently distributed throughout the Neotropical region, The worldwide decline of ocean fisheries stocks has with a high proportion of detritivorous fish species from provided impetus for rapid growth in fish and shellfish the two families Prochilodontidae and Curimatidae farming, or aquaculture.Between 1987and 1997, (GURGEL et al., 2012).These families include stocks of global production of farmed fish and shellfish (collectively important fish species which account for approximately called 'fish') more than doubled in weight and value, as fifty percent of the community biomass of some regions did its contribution to world fish supplies.Fish produced (BOWEN, 1983;FLECKER, 1996).Additionally, Maia et from farming activities currently accounts for over oneal. (1983) by analyzing the chemical composition of some quarter of all fish directly consumed by humans.As the species of the genus Prochilodus showed the low fat level human population continues to expand beyond 6 billion, and high protein level, and thus, highly important species its reliance on farmed fish production as an important for fish farming.Among the endemic freshwater fish, source of protein will also increase (ROSAMOND, 2000).Biota Amazônia potential for aquaculture, in Brazil the Prochilodontidae maturation (VAZZOLER, 1996).The extraction of the family is important, which Prochilodus brevis, regionally gonads was performed through longitudinal abdominal known as curimatã, is part of (SAINT-PAUL, 1986).It is a incision, then fixed in Bouin's solution and submitted to rheophilic fish which migrates during the rainy season for histologic treatment by techniques of Hematoxylin-Eosin several kilometers to the headwaters of the river to spawn (MICHALANY, 1990).(CHELLAPPA et al., 2009).Although it has considerable economic importance in northeastern Brazil, its reproductive Data analysis biology is scarcely known (ARAÚJO et al., 2003;GURGEL The scale of gonadal maturation was based on et al., 2012;NASCIMENTO et al., 2012).macroscopic and microscopic characteristics (VAZZOLER, The cultivating activity requires handling and creation 1996) and average values of the Gonadosomatic Index measures that can be improved through studies about (GSI).The age at maturity was predicted with a satisfactory reproduction in fish.In this sense, the knowledge of degree of confidence, because the gonadal development reproductive tactics is fundamental for the comprehension of was observed from fingerlings until adulthood.the cycle of life strategies as well as to guide measures of The beginning of the reproductive activity was administration, handling and preservation in front of determined using the distribution of percentage frequency impacts, as the exhaustion of the natural resources of the individuals by stages of gonadal maturation.The (VAZZOLER, 1996).This way, the knowledge of the curve of gonadal maturation was established based on morpho-histological characteristics of the gonads´ structures average values of the Gonadosomatic Index (GSI) with is a basic and primordial stage for the comprehension of regard to the age in days post-hatching of fishes, according fish reproduction, especially when evaluated since the first to the expression: GSI = Wg/Wt x 100, where Wg is stages of life.
gonad weight and Wt is total weight (WOOTTON et al., Knowing about these reproductive characteristics helps 1978).to improve procedures which enable the gonadal For statistical analysis it was used the program Statistica maturation in captivity, so the induction of spawn can be 10.0 to verify, through the methods of analysis of variance performed (CONSTANTINOS et al., 2010).This becomes (one-way ANOVA) and Fisher, the existence of significant valid in front of reports about inadequate or insufficient differences, at the level of 5%, among the average values results obtained with the reproduction induced with of the variables GSI , with regard to the stages of gonadal hormones, many times due to inadequate handling of the maturation and age, in days post-hatching, of the fishes.gametes in distinct stages of development, affecting their quality; the developmental stage of the gonads at the time 3. Results the hormonal therapy is applied; the type of hormonal Prochilodus brevis did not exhibit any secondary sexual therapy; the possible stress induced by the manipulation characteristics during the reproductive period, and hence no necessary for the hormone administration and, in the case of sexual dimorphism was observed.As such, it was possible to artificial insemination, the latency period between hormonal identify the sex of each individual only after extraction of stimulation and stripping for in vitro fertilization the gonads performed through a longitudinal abdominal (CONSTANTINOS et al., 2010).
incision.The following macroscopic stages of gonadal Therefore, this study aims to analyze the reproductive maturation were identified for both sexes: immature, biology of Prochilodus brevis in captivity since hatching of maturing, mature and regression (Figure 1). the fingerlings, to identify the age of the fish in that each stage of gonadal maturation takes place.And thus contribute to the conservation and improvements in procedures for the maturation in captivity.

Material and methods
The fish larvae were obtained at Estevão de Oliveira Fishery Station, in Caicó/RN/Brazil.After ninety days of hatching of fingerlings, these were transferred to a tank measuring 9x3x1.5 meters, previously prepared with addition of land in the Agricultural School of Jundiaí in the city of Macaíba/RN/Brazil.The feeding, with industrial fish food, happened twice a day.
With the use of cast nets, with mesh 2 x 2 cm and length of 2.50 meters, were captured a total of 137 specimens of P. brevis since the early stages of life to adulthood.Sampling was fortnightlt, in the period of July 2010 to April 2011, between 8:00h am and 10:00h am.
Of each fish were registered data of total length and standard length, in centimeter, total weight (Wt) and weight of the gonads (Wg), in grams, sex and stage of gonadal  represent immature; (c) and (d) maturing; (e) and (f) mature; (g) and (h) regression.Scale bar= 2 cm.Biota Amazônia

Ovarian development
Testicular development For Prochilodus brevis in captivity, the development of For males, it was verified that the development of the testicles of P. brevis in captivity is processed at four stages: the ovaries was processed in four stages: Immature, In Immature, In Maturation, Mature, and Regression.Maturation, Mature and Regression, described below: Stage I: Immature -Testicles are very thin, filiform and Stage I: Immature -Immature ovaries are characterized translucent macroscopically.When observed in light macroscopically for showing translucent pink color, being microscope, show abundant interstitial stroma, few flattened dorsoventrally and small, occupying a small spermatogonias distributed randomly and absence of portion of the abdominal cavity and showing little blood apparent seminiferous tubules (Fig. 3a).supply .Microscopically (Figure 2a), they reveal oocytes at Stage II: In Maturation -Testicles are broader, assuming stage I (chromatin-nucleoli) and at stage II (perinucleolar), pinkish white color.In the light microscope, the seminiferous with predominance of this last one, inserted in the ovigerous tubules show cysts of different phases of the lamellae.At this stage are included females entering in spermatogenic lineage cells and lumen with low quantity of gonadal maturation for the first time.
spermatozoids (Fig. 3b).Stage II: In Maturation -The ovaries are bigger, of Stage III: Mature -Testicles reach their maximum greyish-green color, with central blood vessel and intense development, with shape of tumescent strings and dense, peripheral vasculature.They occupy a greater portion of with pink or milky white color, able to perform the the abdominal cavity compared to the previous stage.
spermiation.Histologically, they show high amount of Histological sections evidence oocytes at phases II, III, IV spermatozoids occupying the lumen of the seminiferous and V (Figure 2b), which abundance depends if the ovary tubules and spermatic duct (Fig. 3c). is at phase of initial, intermediate or final maturation.
Stage IV: Regression -The germinal epithelium, Stage III: Mature -Ovaries occupy the whole discontinuous, shows reminiscent vacuolated cysts and the abdominal cavity, pressing the viscera and determining the interstice is thick.The tubular light appears, irregularly, with animal´s distension of the abdomen.The color of the spermatozoids (Fig. 3d).oocytes is light green.At this stage, ovaries are richly These results show that P. brevis in captivity reaches all vascularized.Histological sections reveal total stages of gonadal maturation naturally until the mature predomination of oocytes at phase V (Mature) and some stage.In mature stage, oocytes in the final stages of oocytes at phase II (perinucleolar) (Figure 2c).development (V oocytes) initiate follicular atresia, Stage IV: Regression -Ovaries with oocytes distributed interrupting spawning.However, as follicular atresia was randomly, white and pink.The vascularization becomes not observed in oocytes reserve (II oocytes), then the next congested, showing evidences of the phase of follicular reproductive cycle occurs the same way.atresia.As the regression process advances, the amount of white and pink oocytes increases as well as the hemorrhagic aspect.Histologically, it is observed oocytes at the phases of initial (Figure 2d), intermediate and final atresia.

Variation of the Gonadosomatic Index
The average values of gonad weight (Wg), gonadosomatic index (GSI), with the respective standard deviations (s.d.), for females and males are shown on Table 1.days post-hatching (dph), with presence of individuals in "mature" stage from 274 dph, reaching totality at 295 dph.The "regression" stage began from 359 dph (Figure 6).
Both for females and males, the highest values of gonadosomatic index (Figure 4) happened at the stages "in maturation" and "mature", significantly differing from the stages "immature" and "regression" (p= 0,000).The variation of the maturation curve based on the average For the males, "in maturation" stage began at 247 dph, values of the gonadosomatic index was consistent with the and the appearance of mature individuals at 274 dph macroscopic and microscopic observations of the gonads.
reaching the totality at 359 dph.About the "regression" stage, it was found from the age of 410 dph (Figure 7).

Stages of maturation
Gonadosomatic Index by days post-hatching of the The distribution of average values of length and females (Figure 7) showed elevation from 247 dph, with weight (Figure 5) by age in days post hatching (dph) of maximum values at 340 dph.About the distribution for fishes showed higher averages in the period from 247 the males, it was noticed more elevated values between dph to 359 dph, befitting the period of maturation of the 324 and 359 dph (Figure 8).gonads (Figures 5, 6).
The frequencies of the different stages of maturation on the 4. Discussion sampled individuals differed between the sexes.For the females, Structural changes along the reproductive cycle was presented higher frequency of "in maturation" stage at 247 enable inferring about the functional state of the gonads.The description of gonadal development, based on ready for the spawn that are not submitted to any macro and microscopic observations, is being largely hormonal treatment, suffering normal ovarian regression.used in the determination of the reproductive period of In this study, it should be pointed out that the presence of the species (BARBOSA et al., 2012).
the atretic follicle was observed only in mature ovaries, in the final stages of oocyte maturation (mature oocyte), According to Bazzoli (2003), in Characiformes the never in previous phases of maturation, therefore morphological characteristics observed in the ovaries like differing from the observations performed by Rizzo and volume, color and thickness suffer seasonal modifications, Bazzoli (1995), which in females of Prochilodus affinis, according to the stages of the reproductive cycle, just like it kept in tanks, the degenerative phenomena can happen at was verified for the gonads of Prochilodus brevis.The any stage of oocyte maturation.referred author affirms that the color of mature ovaries depends on the color of their vitelogenic oocytes.For the According to Romagosa et al. (1985), the oocytes of fishes of River São Francisco hydrographic basin are Piaractus mesopotamicus at stage of ovarian regression considered three predominant categories of colors: a) are similar to the ones of curimbatá, when opaque and yellow, in Tetragonopterinae, Erythrinidae, Curimatidae, white oocytes are observed.In the present study were also Doradidae, Pimelodidae and Loricarridae; b) gray, in identified opaque and white oocytes of P. brevis at the Anostomidae and Prochilodontidae and, c) green, stage of ovarian regression.Bryconinae, Salminae and Pseudopimelodus.In the present In contrast to the regression process in captivity, the study, it was observed mature ovaries of greyish green technique of "hipofization" is being widely used to induce color.
reproduction in migratory fish through the application of In most Characiformes, testicles are paired organs, natural hormones present in the hypophysis of mature fish.stretched and fusiform, located dorsally-laterally to the However, the regression period can affect the artificial swim bladder and coelomic cavity and dorsally to the reproduction of the species since degenerative processes digestive tube.They are attached to the swim bladder in the ovaries reduce the rate of fertilization (RIZZO; through the mesorchium (BAZZOLI, 2003).BAZZOLI, 1995).Therefore, knowledge about the process The testicles of curimatã are paired organs, free in the of gonadal regression as well as the age at which the anterior and middle portions, linked in the third posterior species begins the process of follicular atresia in captivity forming a unique spermatic duct; they have smooth helps the new methods of artificial reproduction.external surface and suffer modifications in volume, shape It is also possible to stimulate the reproduction of and color during the gonadal development, like migratory fish through environmental induction, triggering Nascimento et al. ( 2012) demonstrated, such observations the entire process in captivity by maintaining environmental corroborate the findings in the present study for conditions suitable to stimulate reproduction.However the Prochilodus brevis.The morphological variations observed complexity of the environmental mechanisms that control in the testicles of P. brevis, during the reproductive process gonadal development and reproductive behavior makes it are related to the stages of gonadal maturation, being very difficult to simulate in a situation of captivity.Results limited the changes in color (from transparent in immature that prove the success of the environmental induction for the phase to milky white at the reproductive period), shape, final maturation and spawn of the Brazilian migratory fish volume and vascularization.
are very rare (FILHO; WEINGARTNER, 2007).According to Agostinho et al. (2003), piracema In this study, the average values of the Gonadosomatic (Brazilian phenomenon of migration) fish, known as Index (GSI) were higher for female when compared to rheophilic, need to migrate at the reproductive period, to male´s values.These results indicate that the increase in promote the final maturation of the gonads.According to volume of the ovaries during the maturation process the authors, the period of floods, the increase of the happens in a more accentuated way than for the testicles, photoperiod, the amount of dissolved ions (conductivity) which is caused by the accumulation of yolk vesicles by and the water´s temperature are the factors that induce those ones.The distribution about the stages of gonadal the finalization of this process.Fishes which show this maturation showed the highest values for stages "in behavior only release their gametes under these starting maturation" and "mature" and the lowest values for signals; otherwise, the spawn does not happen even if the specimens in "regression", considering fishes in gonads are already developed.In this context, as P. brevis reproductive activity.Araújo et al. (2003) found similar is a rheophilic species, it was observed the process of results for Prochilodus cearensis.These results demonstrate follicular atresia under captivity situation.According to that Gonadosomatic Index is good indicator of Filho and Weingartner (2007), the fact of a species not reproductive activity of fish.performing reproduction in captivity, during the fattening From the results it was possible to evaluate that the phase, can be considered an advantage, because it curimatãs held in captivity showed the average values of enables that the energy supplied in food can be canalized body measurements (length and weight) consistent with the to the growth of the body, instead of directed for the growth and maturation of gonads.This can be explained gonadal development and reproductive behavior.
because during the maturation gonadal, the fish require The histologic analysis of ovaries of P. brevis, when kept larger body proportions to accommodate the gonads and in confinement, showed that the processes of follicular thus ensure greater fecundity (VAZZOLER, 1996).atresia, or oocyte degeneration, happen in specimens

Figure 6 .Figura 7 .Figure 8 .
Figure 6.Relative frequency of the stages of gonadal maturation by days post-hatching of curimatã fish, Prochilodus brevis, for females.

Table 1 .
Average values of gonad weight (Wg), gonadosomatic Index (GSI), with the respective standard deviations, for females and males of Prochilodus brevis.