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Studies on Reproduction and Larval Rearing of Amazonian Fish 10NSR2 and 2A

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Studies on Reproduction and Larval Rearing of Amazonian Fish

New Aquaculture Systems/New Species Research 2 and 2A (10NSR2 and 2A)/Experiment/Peru

Collaborating Institution
Instituto de Investigaciones de la Amazonia Peruana, Peru
     Fernando Alcántara
     Salvador Tello

Southern Illinois University at Carbondale
     Christopher C. Kohler

The Ohio State University
     Mary Ann Abiado
     Konrad Dabrowski

Objectives
1) Evaluate and compare growth performance of gamitana, Colossoma macropomum, and paco, Piaractus brachypomus, larvae fed different feeds.

2) Determine changes in plasma sex steroid hormones during an annual cycle and those preceding ovulation and spermiation in two Amazonian catfishes, Pseudoplatystoma fasciatum and P. tigrinum.

Significance
Gamitana, Colossoma macropomum, and paco, Piaractus brachypomus, are two important aquaculture species in South America (Saint-Paul, 1992). These Characidae have a commercial value because of their high growth rate and the quality of their flesh (Saint-Paul, 1992; Vieira and Johnston, 1996). During the Ninth Work Plan, we successfully induced ovulation and spermiation of P. brachypomus using LHRHa. Both genders were injected with two doses of LHRHa. The concentration of preparation was 0.0042 mg of equivalents of active hormone per ml. Males and females were injected with 1 ml/kg and 2.6 ml/kg, respectively. The priming dose (50% and 10% in males and females, respectively) was administrated in the morning, whereas the resolving dose (50% and 90% in males and females, respectively) was injected at 2200 h. Oviposition was observed within 8 to 16 hours following the resolving dose of the hormone and survival at 13 hours of incubation amounted to 68.5 ± 25%. Our challenge now is to rear these larvae in intensive culture using dry food. This is important because survival of larval paco stocked directly to ponds was very low in IIAPs experience. The information on the first feeding of Characidae is scarce. Recently, a feeding experiment was carried out to determine the relationship between the live Artemia feeding levels and growth rate in pre-weaning C. macropomum larvae (Sevilla and Gunther, 2000). However, Canzi et al. (1992) and Yamanaka (1988, in Canzi et al., 1992) reported that artificial diets are readily accepted by P. mesopotamicus. Therefore, we propose to investigate the potential for first feeding C. macropomum and P. brachypomus using commercial and experimental diets. Preliminary experiments in 2000 carried out by the authors confirmed the acceptance of formulated diets and growth of paco larvae.

The second objective of this study is focused on the controlled reproduction of two catfish species Pseudoplatystoma fasciatum and P. tigrinum, which are of interest as new aquaculture species in South America (Kossowski, 1996). In Peru, spawning of both species occurs in February-March (Alcantara, personal communication). In P. fasciatum, the oocyte size was used to evaluate the maturity of the gonads and a diameter of 1.8 mm indicated the readiness of the gonads (Kossowski, 1996). Final maturation and ovulation was achieved in several catfish species from South America using carp pituitary extracts or pituitary hormones (Cardoso et al., 1995; Kossowski, 1996). However, to the best of our knowledge, no information is available on the profiles of plasma sex steroids in both target species and we could possibly use this information to synchronize ovulation/spermiation in these fish (Dabrowski et al., 1996). The annual changes in the blood plasma steroids as well as the surge preceding the spermiation and ovulation (maturational hormones) can contribute to a better understanding of the dynamics of gonadal steroidogenesis. Moreover, such information will be useful in the development and standardization of breeding techniques through the use of natural and/or synthetic hormones. Preliminary data indicated that the level of estradiol-17a and testosterone in females of P. fasciatum raised in a pond at IIAP in March averaged 0.35 ± 0.2 ng/ml and 3.18 ± 2.5 ng/ml (n = 4).

Quantified Anticipated Benefits
The proposed study aims to investigate key aspects of nutrition and reproduction biology of several Amazonian freshwater fish species such as C. macropomum, P. brachypomus, P. fasciatum, and P. tigrinum in order to improve or develop sustainable aquaculture technology for these species.

Through our collaborative effort with Peruvian investigators we will be able (1) to develop the procedures of first-feeding of C. macropomum and P. brachypomus, and (2) to monitor and understand the dynamics of gonadal steroidogenesis during maturation of P. fasciatum and P. tigrinum. These data on steroid profiles will be correlated with the quality of gametes produced. Therefore, the first beneficiaries of this research will be the local producers of Colossoma and Piaractus species in the Peruvian Amazon. In our experience, larval gamitana and paco frequently experience low water levels in nursery ponds and high water temperatures. Development of the technology of intensive growth of these species and stocking 4­6-week-old juveniles will dramatically increase their survival and efficiency of production.

Catfish could be cultured in mono- or polyculture systems in order to control the native cichlids like Cichlassoma and Aequidens. Both species reach large sizes (20-40 kg) and have a wide distribution throughout the Amazonian basin in South America (Colombia, Venezuela, Brazil), so experiences gained with these species will be applicable in many countries of the region. P. tigrinum is an attractive species for the aquarium business, so development of aquaculture technology will reduce pressure on natural stocks and create an additional source of income for local fish farmers.

This study will also contribute towards institutional strengthening by providing training for IIAP staff on various aspects of fish nutrition and reproduction.

Research Design
Location of Work: The experiments will be accomplished at the field station of the Instituto de Investigaciones de la Amazonia Peruana. Additional experiments with the experimental diets and radioimmunoassay analysis will be carried out at the School of Natural Resources, The Ohio State University, Columbus, Ohio, USA.

Methods

Objective 1: Growth Performance of Gamitana C. macropomum and Paco P. brachypomus Larvae

Larvae of both species obtained from induced reproduction will be used for the feeding experiment. The feeding trial will be conducted in a flow through system consisting of 12 cylindrical tanks (3 tanks/dietary treatment) supplied with aeration. Water quality will be monitored throughout the larval rearing process. Temperature (26-28°C) and dissolved oxygen (5-6 mg/L) will be determined on a daily basis with biweekly measurements of total ammonia-nitrogen and pH. Two days after yolk resorption, larvae will be randomly distributed at a density of 5,000 larvae/tank and fed at a restricted ration up to 90% satiation for 2-4 weeks. At the beginning of the experiment, 30 larvae will be weighed and frozen for subsequent analysis of whole body composition. Larvae will be fed 4 diets: 1) live food (Artemia nauplii), 2) a commercial diet (Kyowa Hakko Kogyo Co., Ltd., Japan), 3) an experimental casein-gelatin­based diet (Lee et al., 2001) and 4) an experimental lecithin based diet (Infante and Cahu, 1999). Both experimental diets will be formulated based on our previous experience and will be isonitrogenous (protein requirement: 40-55% for most larval fish, Dabrowski, 1986). Larval samples (n = 20) will be taken every week from each tank and fixed in buffered formalin for biometric measurements. At the end of the experiment, growth performance will be evaluated in terms of final individual body weight, survival (%), specific growth rate (SGR, %) and weight gain (%). Fish from each dietary treatment also will be sampled for proximate analysis (water, protein, lipid, ash).

Objective 2: Changes in Plasma Sex Steroid Hormones in P. fasciatum and P. tigrinum

Broodstock fish from both species will be raised in ponds at the IIAP. Water temperature and dissolved oxygen will be recorded daily. At the beginning of the gonad recrudescence, twenty-five fish of both species will be captured, individually measured, weighed and tagged (PIT-tags, Biosonic, Seattle, WA). Fish will then be released into their respective ponds. Blood will be collected every 3-6 months from the caudal vessel of unanesthetized fish using a heparinized syringe. Blood will centrifuged at 1,500 g for 15 min and the plasma stored at ­20°C until assays. At the time of maturation (normal spawning time in Peru is late February to early March), spermiating males and robust females will be selected. Ovarian maturity will be assessed with a microscope using oocytes collected from the ovary with a catheter. Pairs of catfish will be moved into indoor 0.75-m3 concrete tanks. In each tank, the male will be separated from the female by a net. Intensive aeration of the water will be provided upon fish arrival. Both genders will be injected with two doses of LHRHa to induce ovulation and spermiation. Blood will be collected prior to the priming injection and after ovulation or spermiation as described previously (Dabrowski et al., 2001). Semen and ovules will be collected by stripping after anesthesia (Cardoso et al., 1995). Sperm concentrations will be estimated microscopically using a Double Neubauer Counting Chamber and then spectrophotometrically to develop an appropriate equation (Ciereszko and Dabrowski, 1993). The sperm motility will be evaluated by the same observer and recorded as percent motile sperm. Time of ovulation, fecundity and egg size will be recorded in all females. To test the quality of the eggs, ovules from individual females will be fertilized with pooled semen from three to four males and incubated in separate vertical (conical) incubators of 40 1 capacity. The rate of survival will be assessed at hatching (19 h at 25°C). Hatching success of the embryos will be calculated and expressed as a percentage of the initial number. The plasma concentrations of steroids (testosterone, estradiol-17a, 11-ketotestosterone, and 17,20b-dihydroxy-4-pregnen-3-one) will be measured by radioimmunoassay similar to those used previously (Ottobre et al., 1989) following ethyl-ether extraction. Validation of those steroid assays has already been accomplished in our laboratory for rainbow trout (Dabrowski et al., 1995), paco (Dabrowski et al., 1997), yellow perch (Ciereszko et al., 1997), lake whitefish (Rinchard et al., 2001), and muskellunge (Dabrowski et al., 2000). Assays for those four steroids will be validated in a similarly rigorous fashion for the two target species.

Statistical Analysis: Analyses will be performed using the Statistical Analysis System (SAS Institute, Inc., Cary, NC). Data on growth performance, survival, and plasma sex steroid levels will be subjected to one-way analysis of variance (ANOVA) followed by a comparison of means using Scheffe's F test (Dagnelie, 1975). Normality and homogeneity of variance tests will be performed on raw data. Sample distributions violating assumptions will be log-transformed before analysis. Data, expressed as percentages, will be arc sine-transformed before analysis. All differences will be regarded as significant at P < 0.05. Individual tagging of broodfish will allow repeated blood sampling and observation of the evolution of steroid hormones throughout the annual cycle.

Regional Integration
Research efforts being proposed are logical initial steps toward developing sustainable aquaculture in the region. The research will benefit the entire region by providing pertinent information on broodstock reproduction and nutrition. Terra Nuova is the local organization involved in construction and help in local farmer community. Alcantara will be involved in dissemination of both fish produced during this project and information related to production technologies.

Schedule
Objective 1: Growth Performance of Gamitana C. macropomum and Paco P. brachypomus Larva
July-August 2001, formulation and preparation of the experimental diets
September-October 2001, preparation of the spawning and larval rearing tanks
November-December 2001, induction of spawning of both target species using LHRHa
December 2001-February 2002, feeding experiments for 2-4 weeks duration
February 2002, collection of data on growth and survival
April-December 2002, data analysis and preparation of reports and publications

Objective 2: Changes in Plasma Sex Steroid Hormones in P. fasciatum and P. tigrinum
July 2001-December 2002, collection of blood samples every 3-6 months
January 2002, preparation of the spawning tanks
February-March 2002, induction of spawning of both target species using LHRHa, collection of blood samples prior to the priming injection and after ovulation or spermiation, evaluation of sperm and egg quality
April-December 2002, analysis of plasma sex steroid hormones by RIA
July-December 2002, data analysis and preparation of reports and publications

Final Report: 30 April 2003

Literature Cited
Canzi, C., J.R. Borghetti, and D.R. Fernandez, 1992. The effects of different treatments on the survival and development of pacu larvae (Piaractus mesopotamicus). Arq. Biol. Tecnol., 35:117­127.

Cardoso, E.L., M.S. Alves, R.M.A. Ferreira, and H.P. Godinho, 1995. Embryogenesis of the neotropical freshwater siluriforme Pseudoplatystoma coruscans. Aquat. Living Resour., 8:343­346.

Ciereszko, A. and K. Dabrowski, 1993. Estimation of sperm concentration of rainbow trout, whitefish and yellow perch using spectrophotometric technique. Aquaculture, 109:367­373.

Ciereszko, R.E., K. Dabrowski, A. Ciereszko, J. Ebeling, and J.S. Ottobre, 1997. Effects of temperature and photoperiod on reproduction of female yellow perch Perca flavescens: plasma concentrations of steroid hormones, spontaneous and induced ovulation, and quality of eggs. J. World Aquacult. Soc., 28:344­356.

Dabrowski, K., R.E. Ciereszko, J.H. Blom, and J.S. Ottobre, 1995. Relationship between vitamin C and plasma testosterone in female rainbow trout, Oncorhynchus mykiss. Fish Phys. Biochem., 14:409­414.

Dabrowski, K., J. Rinchard, R. Ciereszko, A. Ciereszko, and J. Ottobre, 1997. Observations on maturity and spermiation of the Amazonian fish Colossoma sp. in North American. Abstract in Biology of Tropical Fishes, Manaus (Brazil), October 6­8, 1997, p. 127.

Dabrowski, K., J. Rinchard, F. Lin, M.A. Garcia-Abiado, and D. Schmidt, 2000. Diploid gynogenetic muskellunge induced with irradiated sperm of yellow perch. Is muskellunge male homogametic? J. Exp. Zool., 287:96-105.

Dabrowski, K., R.E. Ciereszko, A. Ciereszko, G. Toth, S. Christ, D. El-Saidy and J.S. Ottobre. 1996. Reproductive physiology of yellow perch (Perca flavescens): Environmental and endocrinological cues. J. Appl. Ichthyol., 12:139­148.

Dabrowski, K., J. Rinchard, J. Ottobre, F. Alcantara, P. Padilla, A. Ciereszko, M.J. De Jesus, and C. Kohler, 2001. Effect of oxygen saturation in water provided to broodstock and embryos of Piaractus brachypomus on viability of larvae. (in preparation)

Dagnelie, P., 1975. Théorie et Méthodes Statistiques. Applications Agronomiques. Volume II: Les Méthodes de l'Inférence Statistique. Les Presses Agronomiques de Gembloux, Gembloux, Belgium.

Infante, J.L.Z. and C.L. Cahu, 1999. High dietary lipid levels enhance digestive tract maturation and improve Dicentrarchus labrax larval development. J. Nutr., 29:1195­1200.

Kossowski, C., 1996. Perspectives de l'elevage des poissons-chats (Siluroidei) en Amerique du Sud. Aquat. Living Resour., 9, 189­195.

Lee, K.J., K. Dabrowski, J. Rinchard, C. Gomez, and C. Vilchez, 2001. The effects of maca meal on growth and sex differentiation of juvenile rainbow trout. Abstract in Aquaculture 2001, Lake Buena Vista (Florida, USA), January 21­25, 2001, p. 361.

Ottobre, J.S., B.S. Houmard, and A.C. Ottobre, 1989. Luteal production of steroids and prostaglandins during stimulated early pregnancy in the primate: differential regulation of steroid production by chorionic gonadotropin. Biol. Reprod., 41:393­400.

Rinchard, J., K. Dabrowksi, and J. Ottobre, 2001. Plasma sex steroids in lake whitefish Coregonus clupeaformis during spawning in lake Erie. Comp. Physiol. Biochem. C. (in press)

Saint-Paul, U., 1992. Status of aquaculture in Latin America. J. Appl. Ichthyol., 8, 21­39.

Sevilla, A. and J. Gunther, 2000. Growth and feeding level in pre-weaning tambaqui Colossoma macropomum larvae. J. World Aquacult. Soc., 31:218­224.

Vieira, V.L.A. and I.A. Johnston, 1996. Muscle development in the tambaqui, an important Amazonian food fish. J. Fish Biol., 49:842­853.

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