Induced spawning of the endangered Neotropical species Steindachneridion parahybae ( Siluriformes : Pimelodidae )

The “surubim do Paraíba” (Steindachneridion parahybae) is a freshwater catfish endemic to the Paraíba do Sul River basin, Brazil. This species has been seriously threatened by environmental disturbances in the last several decades. Wild Steindachneridion parahybae males and females were collected in 2003 and taken to the hatchery of a power plant of the Companhia Energética de São Paulo (CESP). Steindachneridion parahybae broodstocks were artificially induced to reproduce in December 2003 using a combination of carp pituitary extract (CPE) and human chorionic gonadotropin (hCG). Oocytes and milt were stripped; the fertilized eggs were transferred to 60-liter conical incubators and hatched larvae distributed in nine horizontal trays. Exogenous feed was started just after yolk sac absorption. A high rate of cannibalism and photophobia were observed during the larval period, resulting in a 26% survival rate from larvae to fingerlings.

The surubim do Paraíba species is critically threatened (Brasil, 2004;Caneppele et al., 2008;São Paulo, 2008;Honji et al., 2009), and presently, there are no records of this species in the southern rivers of the Paraíba do Sul basin (São Paulo, 2008).A few populations still remain in northern tributaries, but their survival faces constant threats from pollution, river damming, sand extraction from riverbeds and floodplains, predatory fishing, and the introduction of exotic fish species (Hilsdorf & Petrere, 2002).The biological information that is available about the surubim do Paraíba is very scarce.Its spawning season occurs from late November until March, and its feeding habit is mainly carnivorous (ichthyophagous).Their endangered status (Rosa & Lima, 2005) makes any biological study of surubim do Paraíba extremely laborious, especially during sample collection stages.Therefore, the present difficulty of capturing the species may contribute to the paucity of information about this species, which is at risk of becoming extinct without being fully described.
In the past five years, this species has been included in the conservation program undertaken by the Companhia Energética de São Paulo (CESP, 2006).The first goal of the program was to develop an artificial reproduction protocol that would be applied to S. parahybae fingerling production.The objective of this study was to present the results of the first reported artificial spawning of S. parahybae.
Information from local artisanal fishermen made it possible for us to locate a population that still survives in a small tributary of the Paraíba do Sul River.During 2003, fifteen individuals were caught in the Paraíba do Sul River, near the town of Rio das Flores in the state of Rio de Janeiro (22°13'54"S 43°25'15"W), and brought to the CESP hatchery in order to start the first trials of induced hormonal reproduction.The broodstock (100% survival) were maintained in 200 m 2 ponds and fed commercial food (40% crude protein, Purina TC 40).
In December 2003, the animals were selected according to the typical morphological characteristics of sexual maturity (Leonardo et al., 2004), and based on these characteristics, two females and two males were chosen for artificial reproduction.The females were selected by external characteristics according to the hyperemic genital pore (and swollen abdomen), and the males were chosen according to the white color and high fluidity of milt when the abdominal region was squeezed.The selected broodstock were transferred to the laboratory and kept in 1,000 liter tanks.
The animals were induced to reproduce by combining whole acetone-dried carp pituitary extract (CPE) (Fish Braz) with a protocol proposed by von Ihering & Azevedo (1934, 1936) but using CPE (as proposed for the pimelodid Pseudoplatystoma fasciatum, by Leonardo et al., 2004) and human chorionic gonadotropin (hCG) (Pregnyl -Organon), was performed as follows.Two CPE doses (0.6 mg and 5.4 mg CPE per kg of fish body weight, dissolved in 0.9% sodium chloride solution, considering 0.6 ml in each dose) were used for females, with a 12 h interval between the CPE doses.For males, a single dose was applied at the same time as the females' second dose; this dose contained 3 mg CPE per kg and was also diluted in 0.9% sodium chloride solution.A single dose of hCG was given at the same time as the second CPE application.The hCG concentration used was 2 IU per kg for females and 1 IU per kg for males (Harvey & Carolsfeld, 1993).A summary of the doses used in this protocol is shown in Table 1.

Table 1. Protocol of Steindachneridion parahybae induced reproduction
The use of hCG combined with pituitary extracts has been used in fish species (Thalathiah et al., 1988;Kucharczyk et al., 1997;Zohar & Mylonas, 2001;Leonardo et al., 2004) and has proven to be a very successful and consistent strategy in catfish species (Thalathiah et al., 1988).
After hormone administration, each couple was placed in a 1,000 liter glass tank to facilitate the observation of their sexual behavior during spawning.The time necessary for gamete elimination, counted from the second hormone administration until spawning, was calculated as accumulated thermal units (ATU), or degree-hours.Therefore, the ATU is the sum of the water temperature per hour, from the second hormonal application until spawning (Weingartner & Filho, 2005).After 200 ATUs (average temperature of 24 o C), it was possible to observe an increase in the animals' activity, including aggressive behavior that could harm the partner.This behavior was important to determine when to separate males and females and to strip their gametes in plastic containers.Fertilization was performed by gentle mixing using the "dry" method (von Ihering & Azevedo, 1936).Spawning occurred between 240 and 255 ATU.The ATU value for S. parahybae was similar to other siluriform species, such as Rhamdia quelen, where the ATU value was 220 -240 at 22-27 o C (Baldisserotto & Neto, 2005), Pseudoplatystoma corruscans (200 -280 ATU at 28 o C) (Campos, 2005) and Steindachneridion melanodermatum (240 -280 ATU at 27 o C) (Ludwig et al., 2005).
The estimated number of ovules in S. parahybae was 324 ovules per gram of ovulated ovary mass (calculated by weighing the ovulated eggs and counting subsamples in triplicate).Considering the body weight of both females (Table 1), we can conclude that the number of released ovules ranged from 9,000 to 10,000 eggs kg -1 of female body weight.This value is lower than the number found in R. quelen, in which each kilogram of female body weight corresponds to approximately 216,000 eggs (Baldisserotto & Neto, 2005).Higher numbers of eggs are also found in P. corruscans, which produces between 135,000 and 220,000 eggs kg -1 of female body weight (Campos, 2005) releases a smaller mass of eggs (27 -30 g kg -1 , calculated from Table 1) than does P. corruscans.During the 2007-2008 spawning season, after the beginning of physiological studies on the pituitary-gonad axis of S. parahybae, two females were also stripped using the same protocol described here for hormonal induction, and the amount of eggs released increased to about 45 g kg -1 (data not shown).
After spawning and hydration, the eggs were gently transferred from the plastic containers to the 60-liter conical incubators (built at CESP) (Table 1).In general, S. parahybae eggs were floating, yellowish, measuring ca.190 mm.After an incubation of 42 h at an average temperature of 24 o C, approximately 29,200 hatched larvae from both females were divided among nine horizontal trays (built at CESP; 180 x 40 x 8 cm) (3,200 larvae per tray), where they were maintained for 8 days.The yolk reserve was completely absorbed within the first 24 h, i.e., one day after hatching (DAH).The congeneric species S. melanodermatum showed a shorter egg incubation period, 18 h, but a longer yolk absorption period, 48 h, at 27 o C (Ludwig et al., 2005).
The first feeding started on the 2 nd DAH; cannibalism was observed during larval development, as was heterogeneous larval growth and photophobia.Cannibalism has already been observed in many siluriform species (Luz et al., 2001;Baldisserotto & Neto, 2005;Campos, 2005;Ludwig et al., 2005;Schutz et al., 2008) and is one of the main bottlenecks responsible for reducing larval survival.Reducing the amount of cannibalism during this phase of fingerling production will require specific attention.Different photoperiods were proposed to reduce cannibalism in Steindachneridion scriptum.Schutz et al. (2008) suggested a photoperiod of 14 h of light per day to be the best to achieve a higher survival with good growth.Photoperiod studies with the same species suggest that S. scriptum can easily acclimate to the light, displaying higher growth during the photoperiod with continuous light when compared with darkness (Zaniboni-Filho et al., 2008).
During the incubation period -from the absorption of the yolk sac to the 3 rd DAH -larvae were fed a combination (in the same proportion) of Astyanax spp.larvae and Daphnia spp.For S. scriptum larvae, the best growth rate was achieved using fish larvae (Prochilodus lineatus) as initial feeding (Schutz et al., 2008).Beginning on the 4 th DAH, all larvae were fed a commercial powdered diet containing 55% crude protein (CP), mixed with fish meal in a proportion of 4:1 (commercial diet: fish meal).The larvae accepted the artificial diet, and this transition was considered successful as previously suggested for S. scriptum larvae, in which feed transition was implemented between the second and eighth days of cultivation (Adamante et al., 2007).
On the 8 th DAH, larvae measuring 1.2 cm were transferred to two 200 m 3 earthen ponds provided with partial shade.The same feeding protocol was maintained (commercial powdered diet) to feed the larvae from the 4 th DAH in the laboratory trays to approximately 30 days after hatching in the ponds.Animals were maintained for a further five months in the fish ponds with commercial extruded diet with a pellet size of 26 mm and 45% CP.After five months, there were 7,800 surviving fingerlings, each measuring around 12 cm and weighing 23 g, yielding an estimated 26% rate of survival from the larval to fingerling phase.
This first trial of induced reproduction in S. parahybae is an important step towards understanding the reproductive biology of this endangered species, as well as contributing to its conservation.Captive breeding followed by introduction of captive-bred fish into the environment are techniques used widely to recover endangered fish species (Philippart, 1995;Brannon et al., 2004).The production of S. parahybae fingerlings in captivity on a regular basis will allow the establishment of a more effective re-introduction program, as well as an investigation of its potential for fish farming.The protocol reported is viable for induced spawning in S. parahybae, but other hormones and doses should be investigated.Nevertheless, some basic aspects of the reproductive biology of S. parahybae remain unknown, such as the annual gonad histomorphology, which are important factors for establishing an accurate ovarian development cycle and determining the best month for artificial reproduction.Additionally, the annual variation of the gonadosomatic index and relative fecundity, which is important for predicting egg masses and amounts during spawning, has to be investigated.