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Broodstock Diets and Spawning of Colossoma macropomum and/or Piaractus brachypomus 10FFR2 and 2A

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Broodstock Diets and Spawning of Colossoma macropomum
and/or Piaractus brachypomus

Feeds and Fertilizers Research 2 and 2A (10FFR2 and 2A)/Experiment/Peru

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

Universidad Nacional de la Amazonia Peruana, Peru
     Marina del Aguila

Southern Illinois University at Carbondale
     Christopher C. Kohler
     Susan T. Kohler

The Ohio State University
     Konrad Dabrowski

University of Arkansas at Pine Bluff
     Rebecca Lochmann

1) Compare the effects of isocaloric high (40%) and low (30%) protein diets on reproductive performance of Colossoma and/or Piaractus broodstock as indicated by biochemical composition of plasma, ovules, and semen.

2) Compare spawning performance and annual cycles of blood plasma steroid concentrations for Colossoma and/or Piaractus broodstock fed isocaloric high (40%) and low (30%) protein (anchovy fish meal) diets.

A need exists to evaluate the aquaculture potential of local and native species, and to develop appropriate culture technologies. Colossoma spp., Piaractus spp., and their hybrids are important food fishes in the Amazon Basin. Male and female C. macropomum generally reach sexual maturity in 3 and 4 years, respectively, when they have attained 3 to 6 kg in weight. Piaractus become sexually mature about a year sooner, and at a smaller size (2 to 4 kg). Fish held in captivity must be induced to spawn using hormones (Gonzales, 1987). After hormonal treatment gametes are stripped and mixed using the dry method (Alcantara and Guerra, 1992). Both MacDonald-type and Woynarovich-type incubators are employed, with the latter being most common. Hatching occurs 10 to 20 h post-fertilization at 26 to 29°C.

Research on spawning and grow-out of gamitana Colossoma macropomum and paco Piaractus brachypomus, two important foodfish in Peru, were initiated in the Ninth Work Plan. One of our specific objectives was to determine the changes of plasma sex steroid hormones occurring prior to ovulation or spermiation in P. brachypomus. For this purpose, we induced ovulation and spermiation using LHRHa. The response patterns of plasma sex steroids to the hormonal treatments were similar in both genders. The concentrations of testosterone and 17,20b-dihydroxy-4-pregnen-3-one significantly increased (Dabrowski et al., 2001). However, we observed that the levels reached under normoxic conditions were higher than the ones recorded under hypoxia. Hypoxia resulted also in significantly lower survival of embryos (17.3 ± 28%) in comparison to normoxic conditions (68.5 ± 25%). Therefore, the complete spawning failure observed at the Instituto de Investigaciones de la Amazonia Peruana (IIAP) station in Iquitos during the previous years may be related in part to low concentration of oxygen in the water in which the broodstock are stocked prior to spawning as well as the hypoxia (below 4 mg O2/L) during the egg incubation.

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. In the Tenth Work Plan, we will determine the effect of improved broodstock nutrition on maturation and spawning performance of C. macropomum and/or P. brachypomus.

Nutrition is known to affect reproductive success in fishes (De Silva and Anderson, 1995), but the effects are not well documented in colossomids. Past spawning failures of captive colossomids in Iquitos in the past could be due partly to poor nutrition. The diet used currently to maintain broodstock in Iquitos contains about 32% protein, which is intermediate between the requirements for larval and adult colossomids (Araujo-Lima and Goulding, 1997). However, the diet appears to contain a suboptimal amount of total energy compared to diets used in most feeding experiments with colossomids. Insufficient levels of non-protein dietary energy can cause excessive protein catabolism to meet energy requirements, resulting in loss of essential amino acids for other critical functions such as gamete formation. After spawning, the developing larvae are dependent mostly on proteins of maternal origin to continue normal growth and development until exogenous feeding begins. Therefore, the dietary protein and energy:protein ratios of the broodstock diets could be a major determinant of spawning success and larval quality in colossomids. In this study, reproductive performance of colossomids fed two diets similar in total energy but differing in crude protein content (30 or 40%) will be compared in a field trial to that of broodstock fed the diet currently in use in Iquitos for two spawning seasons. Reproductive performance will be assessed using biochemical data from plasma, ovules and semen of broodstock fed the different diets.

Quantified Anticipated Benefits
This project will result in improved nutrition for broodstock of two important foodfishes in the Peruvian Amazon, Colossoma macropomum and Piaractus brachypomus. Spawning performance is expected to significantly improve for both species as an outcome of the research. The further development of sustainable aquaculture of these two species will benefit many sectors throughout the Peruvian Amazon. Rural farmers will benefit by the addition of an alternative to other forms of agriculture. Aquaculture production will require considerably less land than that needed for cattle ranching. Moreover, ponds can be used year-after-year whereas rain forest lands converted to traditional agricultural practices are rarely productive for more than a couple of seasons, and such lands, once abandoned, usually can no longer support normal jungle growth. Both rural and urban poor will benefit by the addition of a steady supply of high quality protein in the marketplace. Aquaculture of Colossoma and/or Piaractus should relieve some of the fishing pressure on these overharvested, native species. These species have been theorized to play a crucial ecological role in disseminating seeds from the flooded forest. Accordingly, the aquaculture of Colossoma and/or Piaractus could be ecologically as well as economically and nutritionally beneficial to the inhabitants of the Peruvian Amazon.

Research Design
Objective 1: Compare the Effects of Isocaloric High (40%) and Low (30%) Protein Diets on Reproductive Performance of Colossoma and/or Piaractus broodstock as Indicated by Biochemical Composition of Plasma, Ovules, and Semen

Two practical diets will be formulated for colossomid broodstock with ingredients commonly used in fish diets in Iquitos, except that palm oil (4%) will be added to each diet to increase the available energy, carotenoid and alpha-tocopherol levels. Palm oil has been used successfully in Colossoma diets previously (Viegas and Guzman 1998). The diets will also be similar in total calorie content, but will differ in total protein content (30 or 40%). The composition of the diets is shown in Table 1. The proportions of feedstuffs in the diets were manipulated to achieve the desired total protein and energy levels. The diets will be prepared in Iquitos, Peru, and their proximate composition will be verified analytically at the University of Arkansas at Pine Bluff. The amino acid and fatty acid profiles of the diet also will be analyzed.

Table 1

A The calculated energy to protein ratios of the diets with 30 and 40% protein are 10.7 and 8.7 kcal/gram dietary protein, respectively, based on digestible energy values for channel catfish or physiological fuel values (NRC, 1993).

B Total dietary protein levels calculated using analyzed protein content of the individual ingredients.

These two new diets will be compared to the broodstock diet currently in use by comparing the reproductive performance of fish fed the different diets in a field trial (described under objective 2). Reproductive performance for objective 1 will be assessed by measuring and comparing the amino acid and fatty acid composition of the ovules and semen from fish fed the different diets. Lipid class composition of ovules, semen and plasma also will be determined by use of an iatroscan at the University of Arkansas at Pine Bluff using methods described previously (Lochmann et al., 1995). Samples will be collected at the same time as those collected for objective 2. Biochemical data will be compared to literature values and to data gathered for objective 2 of this study to determine correlations among indicators of reproductive success in colossomids.

Objective 2: Compare Spawning Performance and Annual Cycles of Blood Plasma Steroid Concentrations for Colossoma and/or Piaractus Broodstock Fed Isocaloric High (40%) and Low (30%) Protein Diets

Colossoma macropomum and/or Piaractus brachypomus broodstock (whichever species is most available) will be raised in three ponds at the IIAP Quistococha Aquaculture Station. Broodstock in one pond will be fed the standard diet that has been used at the station for several years. Fish in another pond will receive the formulated 30% crude protein broodstock diet, while the fish in the third pond will receive the 40% crude protein diet. Water temperature and dissolved oxygen will be recorded daily for each pond. At the beginning of the gonad recrudescence, twenty-five fish 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 assayed. At the time of maturation (normal spawning time in Peru is November to early January), 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 fish 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 as described in section D (Significance). 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 60 L capacity. Egg production/ fish kg will be measured. 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-17b, 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 Ohio State University 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.

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 nutrition and reproduction. Alcantara will be involved in dissemination of both fish produced during this project and information related to production technologies. This experiment contributes to the regional objective of "further refinements in culture technology for Colossoma and/or Piaractus" stated in the Regional Plan for South America.

All activities will take place from 1 July 2001 through 30 April 2003. The broodstock diets will be formulated and made between 1 July and December 2001. Broodstock blood plasma steroid samples will be collected starting 1 July 2001 on a quarterly basis starting 1 July 2001. Broodfish will be spawned in November and December in years 2001 and 2002 (only 2002 spawn will be with fish fed new experimental diets). A final report will be submitted on or before 30 April 2003.

Literature Cited
Alcantara, F., 1985. Reproduccion inducida de gamitana Colossoma macropomum (Cuvier 1818). Ph.D. dissertation, Universidad Nacional de Trujillo, Peru.

Alcantara, F. and H. Guerra, 1992. Avances en la produccion de alevinos de gamitana Colossoma macropomum y paco P. brachypomus por reproduccion inducida. Publication of the Instituto del Mar del Peru (IMARPE) and Instituto de Investigaciones de la Amazonia Peruana (IIAP), 4:23­33.

Araujo-Lima, C. and M. Goulding, 1997. So Fruitful a Fish. Columbia University Press, New York, 191 pp.

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

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

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 flavescence: plasma concentration of steroid hormones, spontaneous and induced ovulation and quality of eggs. J. World Aquacult. Soc., 28:344-356.

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 America. 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., 287:96­105.

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

De Silva, S.S. and T.A. Anderson, 1995. Fish Nutrition in Aquaculture. Chapman and Hall, London, 319 pp.

Gonzalez, J., 1987. Actividad biologica de extractos hipofisarios de Teleosteos y sus cambios en los ciclos.

Lochmann, S.E., G.L. Maillet, D.T. Frank, and C.T. Taggart, 1995. Lipid class composition as a measure of nutritional condition in individual larval Atlantic cod (Gadus morhua). Can. J. Fish. Aquat. Sci., 52:1294­1306.

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

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

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