aquaculture crsp 21 annual technical...

AQUACULTURE CRSP 21ST ANNUAL TECHNICAL REPORT

85

STUDIES ON REPRODUCTION AND LARVAL REARING OF AMAZONIAN FISH

Tenth Work Plan, New Aquaculture Systems/New Species Research (10NSR2)Final Report

Konrad Dabrowski, Kyeong-Jun Lee, Jacques Rinchard, and Mary Ann G. AbiadoSchool of Natural ResourcesThe Ohio State University

Columbus, Ohio, USA

Fernando Alcántara Bocanegra and Salvador TelloInstituto de Investigaciones de la Amazonia Peruana

Iquitos, Peru

Maria Esther Palacios Universidad Nacional Mayor de San Marcos

Lima, Peru

ABSTRACT

This project has developed the techniques and feed formulations that allow the successful rearing of paco (Piaractus spp.) ju-veniles under intensive conditions. Weaning of paco larvae to artificial diets requires initial feeding with brine shrimp, Artemia nauplii, for 7 to 14 d followed by dry feed, which is administered at regular intervals using automatic feeders. We believe that large-scale implementation of this method in Instituto de Investigaciones de la Amazonia Peruana hatcheries (Iquitos and Pucallpa) will significantly improve the larval survival of paco and other Amazonian fishes. The use of locally available plants such as maca, Lepidium meyenii, and aguaje, Mauritia flexuosa, in diet formulations for Amazonian fish larvae could promote the utilization of indigenous plant products in aquaculture feeds. Moreover, the immuno-stimulatory properties of these plants could boost the general health condition of cultured fish. Our work on the assay of plasma sex steroid hormones of Pseudo-platystoma spp. prior to induced ovulation and spermiation could be used as a quantitative tool for selecting good quality broodstock for artificial reproduction schemes. This would help eliminate the costs of hormones and reduce unnecessary stress to broodstock.

INTRODUCTION

Plants originating from the Peruvian Andes, such as the ancient source maca, Lepidium meyenii (Brassicaceae), and the Amazonian fruit species aguaje, Mauritia flexuosa (Arecaceae), and camu-camu Myrciaria dubia (Myrthaceae), are widely used for human consumption and medicinal purposes due to the high nutritional and antioxidant value and phytochemical content (Leon, 1964; Dini et al., 1994; Obregon, 1998; Moraes et al., 1994). These Amazonian fruits are also feed components of Amazonian characins (Piaractus and Colossoma spp.) in the natural environment (Araujo-Lima and Goulding, 1997).

Roots of maca are an edible part of the plant and nutritionally valued and consumed at elevated altitudes in the highlands of Peru since pre-Columbian periods. Presence of phytosterols and high levels of aromatic glucosinolates [benzyl glucosino-late (glucotropaeolin) and p-metoxybenzyl glucosinolate], ali-phatic glucosinolate (allylglucosinolate or sinigrin), fatty acids, amino acids, saponins, and flavonoids has been attributed as the cause of maca’s positive properties to increase energy and improve fertility (Li et al., 2001; Cicero et al., 2001).

Camu-camu is a globose berry fruit of 1 to 3 cm in diameter, red to purple with 2 to 20 g weight, 50 to 55% reddish pulp,

and 1 to 3 seeds per fruit. It is distributed and grows through-out the western Amazon rainforest, especially in swamps or flooded areas, reaching about 2 to 3 m in height. Our analysis of fruits brought frozen from Iquitos and Pucallpa Stations of Instituto de Investigaciones de la Amazonia Peruana (IIAP) has determined that the whole fruits or pulp of camu-camu has the highest concentration of vitamin C, containing 3,000 to 6,000 mg of ascorbic acid per 100 g, ever found in plant material (Lee and Dabrowski, unpublished). In comparison to oranges, camu-camu provides 30 times more vitamin C, ten times more iron, three times more niacin, twice as much riboflavin, and fifty percent more phosphorus. Camu-camu has an exceptional value that has justified planting in Peru and Brazil for export of “natural vitamin C.” This provides good opportunities for local economies, including the utilization in fish diets, as the source of vitamins and phytochemicals.

The palm of aguaje is a massive tree that can grow to over 100 feet tall in the Amazon rainforest. The fruit of aguaje is a sub-globose “pineapple” of 5 to 7 cm in length, 4 to 5 cm in diameter, brown to dark-red in color, 40 to 85 g in weight, 10 to 21% yellow to red-orange pulp, and 1 to 2 seeds per fruit. The fruits are eaten raw or processed, making a sweet paste used for beverages and ice creams. Fruits and seeds also yield edible oil. The fiber of the palm is used to build the roof of houses.

TWENTY-FIRST ANNUAL TECHNICAL REPORT86 APPROPRIATE TECHNOLOGY RESEARCH 87

The leaf is used in handicrafts. The trunk is used to obtain meal with starch of good quality and is also used in paper process-ing. This native plant is not only important for humans, but it is an essential fruit in the diets of the wild animals from the rainforest.

Dispersal fruits and seeds of tropical plant species, such as aguaje and camu-camu, have also been reported as part of the diet of frugivorous fish of the Amazon; in particular, the native Amazon fishes paco, Piaractus brachypomus, and gamitana, Colossoma macropomum. Aquaculture of these fish species has to be developed at extensive and semi-intensive levels because of their high growth potential mainly in earthen ponds or floating cages (Saint-Paul, 1992). The information about the nutritional requirements of Neotropical freshwater species with economi-cal importance, such as serrasalmids, is still scarce.

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 luteinizing hormone-releasing hormone analog (LHRHa). Both genders were injected with two doses of LHRHa. Concentration of the preparation was 0.0042 mg of equivalents of active hormone per milliliter. Males and females were injected with 1 ml kg-1 and 2.6 ml kg-1, 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 h follow-ing the resolving dose of the hormone, and survival at 13 h of incubation amounted to 68.5 ± 25%. Our challenge now is to rear larvae of paco and gamitana using formulated dry feeds. This is important because survival of larval paco stocked directly to ponds was very low in IIAP’s experience. Informa-tion on the first feeding of Characidae is scarce. Recently, a feeding experiment was carried out to determine the relation-ship between 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 proposed to investigate the potential for first feeding of C. macropomum and P. brachypomus using commercial and experimental diets. Preliminary experi-ments in 2000 that we carried out in IIAP-Iquitos 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 spe-cies in South America (Kossowski, 1996). In Peru, spawning of both species occurs in February and March (Alcántara-Bo-canegra, pers. comm., 2001). 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 ex-tracts 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 synchro-nize ovulation and spermiation in these fish (Dabrowski et al., 1996). The annual changes in the blood plasma steroids as well as the surge preceding spermiation and ovulation (matura-

tional hormones) can contribute to a better understanding of the dynamics of gonadal steroidogenesis, synchronization of controlled reproduction, and improvements in gamete quality. Moreover, such information will be useful in the development and standardization of breeding techniques through the use of natural and/or synthetic hormones. Previous data indicated that the level of estradiol-17α and testosterone in females of P. fasciatum raised in a pond at IIAP in March averaged 0.35 ± 0.2 ng ml-1 and 3.18 ± 2.5 ng ml-1, respectively (n = 4).

METHODS AND MATERIALS

Objective 1: Growth Performance of Gamitana, Colossoma macropomum, and Paco, Piaractus brachypomus, Larvae Fed Different Feeds

Study 1a

Semi-purified casein-gelatin based diets were supplemented with 15% (dry weight) wheat meal (D1 or control) or with one of three other Peruvian native plant meals as follows: 15% camu-camu meal (D2), 15% aguaje meal (D3), and 15% maca meal (D4) (Table 1). Five percent of fish protein concentrate (CPSP 90, Sopropeche, Boulogne-Sur-Mer, France) was added to the diets to enhance palatability of the semi-purified experi-mental diets. The diets were moistened with distilled water and then cold-pelleted into 2.0 mm diameter in size and freeze-dried to have less than 5% moisture. Pellets were ground and sieved to obtain particle sizes according to the size of the mouth of fish. Particle size of 710 μm was used at the first two weeks and size of 1,000 μm until the end of the experimental period.

The feeding experiment was performed with red paco (Piarac-tus brachypomus) juveniles, averaging 2.04 ± 0.03 g of initial weight, at the Aquaculture Laboratory of the School of Natural Resources, The Ohio State University (OhSU). Fish were pur-

Component Experimental Diets(% dry matter)

Control(D1)

Camu-Camu(D2)

Aguaje(D3)

Maca(D4)

Wheat Meal 15.00 -- -- --Camu-Camu -- 15.00 -- --Aguaje -- -- 15.00 --Maca -- -- -- 15.00Soybean Meal 5.00 5.00 5.00 5.00Vitamin Mixture 4.00 4.00 4.00 4.00Mineral Mixture 3.00 3.00 3.00 3.00Vitamin C 0.02 0.02 0.02 0.02Choline Chloride 1.00 1.00 1.00 1.00Cod Liver Oil 5.00 5.00 5.00 5.00Cellulose 2.00 2.00 2.00 2.00Crude Protein 50.67 50.74 49.41 50.06Ash 4.54 4.71 4.36 4.82

Table 1. Composition and proximate analyses of the experimental diets (% dry matter)


chased from a commercial aquaculture farm (Ekk Will, Water Life Resources, Florida, USA). Fish were distributed in a semi-recirculating system of 12 glass rectangular aquaria (40 l) at a density of 20 fish per tank. Three aquaria were randomly as-signed to one of four diets, and the feeding trial was conducted for eight weeks. Fish were fed experimental diets with the feeding rate ranging from 4 to 2.6% of body weight. The fish were fed three times per day at 1000, 1330, and 1700 h, seven days per week. Both feeding rate and feeding frequency were based on previous results tested with C. macropomum, a species very close to Piaractus. Temperature was controlled by thermo-stat and maintained at 27.5 ± 0.1oC. Water quality was kept by an installed biological filter and UV irradiation. Accumula-tion of feces was avoided by siphoning the tanks daily and cleaning them every week. The light/dark cycle was regulated at 12L:12D, and feeding was stopped 24 h prior to weighing and/or sampling every two weeks. During sampling, weight gain was evaluated in each tank and the amount of food was readjusted. At the beginning of the experiment, 16 fish were randomly sampled to determine initial body composition and chemical analysis. At monthly intervals three fish per aquari-um were euthanized to determine the proximate and mineral composition. At the end of the feeding trial, weight gain (WG = final body weight – initial body weight), specific growth rate [SGR = (ln final body weight – ln initial body weight) × 100/d], feed efficiency ratio (FER = weight gain/dry feed intake), protein efficiency ratio (PER = weight gain/protein intake), and net protein utilization (NPU = body protein gain × 100/crude protein consumed) were calculated.

Proximate composition of the whole body and diets was de-termined by standard procedures (AOAC, 1995), and mineral compositions of whole body and diets were determined by the inductively coupled plasma (ICP) emission spectrophotometric method using ARI-3560 Spectrometer (Applied Research Labs, Valencie, California).

Study 1b

We imported 10,000 paco, Piaractus mesopotamicus, as a surro-gate species larvae from Isla Pe Acuicultura, Clorinda Formosa, Argentina. These fish were shipped to us as yolk-sac embryos (3 d after hatching). We divided them into three groups:

a) Group 1, larvae attempted to be weaned to dry feed 4 d after hatching (at first feeding);

b) Group 2, larvae weaned to dry food 13 d after hatching; and

c) Group 3, larvae/juveniles weaned to dry feeds 21 d after hatching.

Larvae from Groups 2 and 3 were fed with newly-hatched brine shrimp, Artemia salina, nauplii prior to feeding with dry feeds. For Groups 1 and 2 larvae, the diets tested were as fol-lows: 1) casein-gelatin-based diet (control; Lee et al., 2001); 2) casein-gelatin-based diet plus 15% maca, Lepidium meyenii; 3) casein-gelatin-based diet plus 5% propolis; 4) casein-gelatin-based diet plus 30% freeze-dried beef liver; and 5) commercial diet from INVE. Diets 2 and 3 contained plant-based products (maca and propolis, respectively) that have been identified to contain phytochemicals such as flavonoids (quercetin) and isoflavonoids and antimicrobial and anti-cancer causing agents (Rosalen et al., 2000; Kumazawa et al., 2002). The commercial diet from Kyowa Hakko Kogyo Co., Ltd., Japan described in

the original proposal was not included because this product was recently banned in the United States.

The feeding experiment with Group 2 larvae was performed with larvae averaging 0.0029 ± 0.004 g of initial weight and 6.4 mm initial length. The experiment took place for 25 d (24 February to 21 March 2003). The diet containing 30% freeze-dried beef liver (Diet 4) was not used for Group 3 larvae. The average initial weight and total length of Group 3 larvae were 0.02 ± 0.01 g and 11.2 ± 2.3 mm, respectively.

Fish were distributed in a semi-recirculation system consisting of 12 40-l rectangular glass aquaria. Fish were fed experimen-tal diets with the feeding rate ranging from 4 to 2.6% of body weight. The fish were fed using an automatic feeding system (Charlon and Bergot, 1992) every ten minutes during the pho-toperiod, seven days per week. Temperature was controlled by thermostat and maintained at 25.7 ± 1.8oC. Water quality was kept by an installed biological filter and UV irradiation. Accumulation of feces was avoided by siphoning the tanks daily and cleaning them every week. The light/dark cycle was regulated at 12h:12D, and feeding was stopped 24 h prior to weighing and sampling. At the beginning of the experiment, 20 fish were randomly sampled to determine initial body composition. At the end of the feeding trial, WG, SGR, and FER were calculated.

Objective 2. To 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

Study 2a. Fish Raised in Peru

Broodstock fish raised in Pond 11 at IIAP (Quistococha Sta-tion) were sampled at two characteristic periods, 14 November 2001 (intensive vitellogenic growth) and 16 April 2002 (post-spawning). Fish were individually measured, weighed, and tagged (PIT-tags, Biosonic, Seattle, Washington). Blood was collected from the caudal vessel of unanaesthetized fish using heparinized syringes. Blood was centrifuged at 1,500 × g for five minutes, and the plasma was stored at –20oC until assayed. The plasma concentrations of steroids (testos-terone, estradiol-17α, 11-ketotestosterone, and 17,20β-dihy-droxy-4-pregnen-3-one) were measured by radioimmunoassay, similar to methods used by Ottobre et al. (1989) following ethyl-ether extraction. [1,2,6,7-3H]testosterone [96.5 Curie (Ci) mmol-1] and [2,4,6,7,16,17-3H]estradiol (141 Ci mmol-1) were purchased from NEN Life Science Products (Boston, Massa-chusetts). [3H]11-ketotestosterone (105 Ci mmol-1) was pur-chased from Amersham Pharmacia Biotech (Arlington Heights, Illinois). [3H]17,20β-dihydroxy-4-pregnen-3-one was a gift from A. Fostier (INRA, Rennes, France). Unlabelled steroids were purchased from ICN Pharmaceuticals (Costa Mesa, Califor-nia), Sigma (St. Louis, Missouri), and Steraloids (Wilton, New Hampshire). The testosterone antiserum was provided by the Institute of Animal Physiology (University of Agriculture and Technology, Olsztyn, Poland), the estradiol-17α antiserum by R.L. Butcher (West Virginia University, West Virginia), the 11-ketotestosterone antiserum by D.E. Kime (University of Sheffield, Sheffield, England), and the 17,20β-dihydroxy-4-pregnen-3-one antiserum by A. Fostier. The characteristics of these antisera have been reported previously (Dabrowski et al., 1995; Butcher et al., 1974; Kime and Manning, 1982; and Fostier


and Jalabert, 1986, respectively).

Study 2b. Fish Raised in Columbus, Ohio

On 7 March 2003, 96 surubim (Pseudoplatystoma sp.) were transferred from the University of Wisconsin (Madison) to our Aquaculture Laboratory (OhSU). These fish will be used for our future work as broodstock fish. Fish are currently raised in 200 l tanks, maintained at 25 to 30°C and located in the green-house of the Department of Plant Biology. On 11 March, thirty fish were individually weighed. Blood samples were taken from the caudal vessel with a heparinized syringe, kept on the ice, and then centrifuged at 5,500 rpm at 4oC. The plasma was stored at –20oC until radioimmunoassay. Fifteen fish were sacrificed, and the gonads and the liver were removed and weighed. Gonadosomatic (GSI) and hepatosomatic (HSI) indices (GSI and HSI, respectively) were caculated as GSI = [(gonad weight × 100)/total weight] and HSI = [(liver weight × 100)/total weight]. The gonads were also fixed in Bouin’s solution for histological examination. The plasma concentra-tions of steroids (testosterone, estradiol-17α, and 11-ketotes-tosterone) were measured by radioimmunoassay as described previously.

RESULTS

Studies 1a and 1b

The proximate and mineral composition of each of the four diets tested are presented in Tables 1 and 2, respectively. After eights weeks of the feeding trial, significant differences (P < 0.05) were found in average final body weight (Table 3). Fish fed maca supplement (D4) exhibited the highest average weight gain (P < 0.05), followed by the control diet (D1), and aguaje (D3) (Table 3). Significantly inferior growth perfor-mance was observed in camu-camu (D2) group (Table 3).

Dietary Treatments

ControlD1

Camu-CamuD2

AguajeD3

MacaD4

MACROELEMENTS(μG/G)

Phosphorus 7,542 7,269 7,191 7,651Potassium 3,928 4,787 5,089 6,726Calcium 5,485 5,227 5,366 6,067Magnesium 175 222 268 269Sodium 3,675 4,525 2,781 2,666

MICROELEMENTS(μG/G)

Manganese 214.7 103.4 110.5 ahIron 80.8 92.3 53.4 53.4Copper 4.5 7.7 4.3 4.3Aluminum 51.0 9.0 9.0 14.0Boron 0.8 0.9 1.4 1.1Zinc 28.1 28.7 28.7 32.4

Table 2. Mineral composition of experimental diets.

Variable Dietary Treatments

Control(D1)

Camu-Camu(D2)

Aguaje(D3)

Maca(D4)

Initial Body Weight (g) 2.06 2.00 2.07 2.02Final Body Weight (g) 16.87 b 4.95 c 15.69 b 22.82 a

PER 3.11 b 1.67 c 3.30 ab 3.41 a

FER 1.57 a 0.84 b 1.63 a 1.71 a

SGR (%) 3.93 b 1.83 c 3.98 b 4.50 a

NPU 21.46 b 8.30 c 26.16 a 26.88 a

Instantaneous Food (%) 0.98 0.50 1.12 1.88

Table 3. Growth and feed utilization parameters of red paco juve-niles fed experimental diets

Note: Data in the same row with the same letter superscripts are not significantly different (P > 0.05).

Sample Dry Matter Crude Protein Ash

Initial 13.04 60.62 b 23.18 a

D1 24.78 56.64 c 11.22 c

D2 19.55 67.16 a 14.66 b

D3 26.64 54. 42 c 9.85 c

D4 26.01 57.77 bc 10.62 c

Table 4. Proximate analysis of whole body tissue of paco (Piaractus brachypomus) (% dry matter) initially and at the end of the experiment after eight weeks (D1, D2, D3, D4).


Mineral(µg g-1)

Dietary Treatments

Control(D1)

Camu-Camu(D2)

Aguaje(D3)

Maca(D4)

MACROELEMENTSPhosphorus 17,417 b 22,991 a 15,188 c 16,345 b

Potassium 9,259 b 12,781 a 9,412 b 9,557 b

Calcium 26,842 b 34,567 a 22,742 c 24,881 b

Magnesium 654 b 1,264 a 697 b 684 b

Sodium 4,734 b 6,242 a 3,876 c 4,012 c

MICROELEMENTSManganese 52.80 a 18.08 c 26.27 b 28.78 b

Iron 51.47 a 37.13 ab 28.30 c 49.93 a

Copper 5.83 4.56 7.45 8.53Zinc 127 b 366 a 122 b 110 b

Aluminum 4.11 6.38 2.57 2.39

Table 5. Mineral composition of whole body of paco (Piaractus brachypomus) after eight weeks of feeding with the experi-mental diets.



At the end of the experiment, feed efficiency ratio (FER) was not significantly different among treatments D1, D3, and D4 (Table 3), although fish fed maca diet showed the best FER at the initial period. When tested at monthly intervals, fish fed maca diet showed a greater instantaneous feed intake (P < 0.05) than other treatments (Table 3). The best protein efficiency ratio (PER) was observed in groups fed D4 and was significantly different (P < 0.05) (Table 3). The lowest PER was exhibited in camu-camu groups (D2) (Table 3). Groups fed maca (D4) and aguaje (D3) exhibited higher values of net pro-tein utilization (NPU) and were significantly different (P < 0.05) from the rest of the groups (Table 3). The lowest NPU was found in fish fed camu-camu (D2), and was also signifi-cantly different (P < 0.05) from the other dietary groups (Table 3).

The whole body protein and ash concentrations were signifi-cantly higher in camu-camu groups (D2) than other dietary groups (P < 0.05) (Table 4). The whole body concentration of minerals is shown in Table 5. Mineral composition of fish fed the camu-camu diet (D2) showed significantly (P < 0.05) higher levels of P, K, Ca, Mg, Na, Zn, and Al in comparison to the rest of the diets (Table 5). However, fish fed D1 showed high levels of Mn (P < 0.05), corresponding to an increased level of Mn in D1. This relationship was not found in the case of iron where dietary levels were also variable (Tables 2 and 5).

Among Group 2 larvae, mean final weight and SGR of control fish were significantly higher (P > 0.05) than fish fed any of the formulated diets (D2 to D5) tested (Table 6). Mean sur-vival among Group 2 larvae was around 50%. Among Group


Control (D1) Propolis (D2) Beef Liver (D3) INVE (D4) Maca (D5)

Mean Final BodyWeight (g)

0.15 ± 0.02 a 0.02 ± 0.01 b 0.02 ± 0.01 b 0.02 ± 0.004 b 0.02 ± 0.01 b

SGR (%) 15.7 ± 0.5 a 7.2 ± 1.1 b 8.1 ± 2.0 b 7.9 ± 0.9 b 6.4 ± 2.0 b

Initial Length (mm) 6.4 a 6.4 a 6.4 a 6.4 a 6.4 a

Final Length (mm) 21.7 ± 1.0 a 12.9 ± 0.8 b 13.3 ± 1.2 b 12.0 ± 1.2 b 12.2 ± 1.3 b

Table 6. Growth and feed utilization parameters of paco larvae fed experimental diets (Study 1b, Group 2) for 25 d. Initial body weight of fish was 0.003 ± 0.001 g.


Control (D1) Propolis (D2) INVE (D4) Maca (D5)

Initial Body Weight (g) 0.03 ± 0.01 a 0.01 ± 0.01 b 0.01 ± 0.001 b 0.02 ± 0.01 ab

Mean Final BodyWeight (g) 0.11 ± 0.06 a 0.04 ± 0.01 ab 0.06 ± 0.02 ab 0.03 ± 0.01 b

SGR (%) 6.75 ± 1.39 ab 8.90 ± 5.42 ab 11.56 ± 2.76 a 4.11 ± 1.21 b

Initial Length (mm) 14.1 ± 1.8 a 9.9 ± 1.8 b 9.4 ± 0.6 b 10.5 ± 0.6 b

Final Length (mm) 19.7 ± 3.6 a 15.9 ± 1.1 a 15.9 ± 2.3 a 15.1 ± 1.1 a

Table 7. Growth and feed utilization parameters of paco larvae fed experimental diets (Study 1b, Group 3) for 18 d.

Larvae Dietary Treatments

Control (D1) Maca (D2) Propolis (D3) Freeze-DriedBeef Extract (D4)

INVE (D5)

Group 1 92.3 ± 3.0 a 55.5 ± 5.0 b 64.4 ± 16.7 b 46.0 ± 12.6 b 58.9 ± 16.8 b

Group 2 54.5 ± 4.3 bc 56.9 ± 4.8 ab 65.4 ± 4.1 a 60.6 ± 2.1 ab 45.8 ± 7.7 c

Group 3 54.1 ± 3.9 a 51.7 ± 10.9 a 52.3 a Not Tested 50.9 ± 1.9 a

Note: Means in the same row superscripted with the same letters are not significantly different (P > 0.05)

Table 8. Survival (recorded at 11, 37, and 39 d after hatching for Groups 1, 2, and 3, respectively) of paco larvae weaned to formulated diets at different times after hatching. Group 1 larvae: attempted to wean 4 d after hatching (at first feeding). Group 2 larvae: weaned 13 d after hatching. Group 3 larvae: weaned 21 d after hatching. The average survival percentages of Groups 1, 2, and 3 larvae are 65.2 ± 20.0, 56.8 ± 8.2, and 52.2 ± 5.8%.

Note: Means in the same row superscripted with the same letters are not significantly different (P > 0.05).


3 larvae, mean final weight and SGR of fish fed Propolis and INVE diets did not differ significantly (P > 0.05) from controls (Table 7). We found that mean survival of Groups 1 to 3 larvae was around 50% (Table 8). However, surviving Group 1 larvae appeared weak and emaciated. This implies that weaning this species to dry feeds at later stages would be preferable. Hence,

we completed two experiments with Groups 2 and 3 larvae.

Studies 2a and 2b

The reproductive characteristics of fish sampled in November 2001 (active vitellogenic growth) are presented in Table 9. Be-

Species N Length(cm)

Weight(g)

T(pg ml-1)

E2(pg ml-1)

11-kT(pg ml-1)

17,20ßP(pg ml-1)

P. fasciatum 6 61 ± 6 1,540 ± 372 296 ± 105 7 ± 16 655 ± 561 5 ± 12P. tigrinum 3 70 ± 1 2,115 ± 671 235 ± 47 67 ± 81 610 ± 334 26 ± 46

Table 9. Size characteristics and reproductive hormone levels of Pseudoplatystoma fasciatum and P. tigrinum captured on 14 November 2001 in Pond 11 at IIAP. T = testosterone, E2 = estradiol-17α, 11-kT = 11-ketotestosterone, and 17,20βP = 17,20β-dihydroxy-4-pregnen-3-one.

Species N Length(cm)

Weight(g)

T(pg ml-1)

E2(pg ml-1)

11-kT(pg ml-1)

17,20ßP(pg ml-1)

P. fasciatum 7 55 ± 5 1.2 ± 0.3 186 ± 80 225 ± 219 30 ± 37 ndP. tigrinum 1 90 6.04 467 542 2 nd

Table 10. Reproductive characteristics of Pseudoplatystoma fasciatum and P. tigrinum captured on 16 April 2002 in Pond 11 at IIAP. T = testoster-one, E2 = estradiol-17α, 11-kT = 11-ketotestosterone, and 17,20βP = 17,20β-dihydroxy-4-pregnen-3-one, nd = not detected.

Species N Weight(g)

T(pg ml-1)

E2(pg ml-1)

11-kT(pg ml-1)

17,20ßP(pg ml-1)

P. fasciatum 5 0.39 ± 0.26 65 ± 18 19 ± 42 113 ± 141 nd

Table 11. Reproductive characteristics of Pseudoplatystoma fasciatum captured on 18 April 2002 at Pucallpa. T = testosterone, E2 = estradiol-17α, 11-kT = 11-ketotestosterone, 17,20βP = 17,20β-dihydroxy-4-pregnen-3-one, and nd = not detected.

Sex N Weight(g)

GSI(%)

HIS(%)

T(pg ml-1)

E2(pg ml-1)

11-kT(pg ml-1)

Female 13 212 ± 74 0.4 ± 0.2 0.6 ± 0.1 208 ± 92 45 ± 22 158 ± 113Male 2 278 ± 61 0.2 ± 0.1 0.7 ± 0.1 234 ± 57 25 ± 1 912 ± 491

Table 12. Reproductive characteristics of surubim sampled on 11 March 2003 at The Ohio State University. GSI = gonadosomatic index, HSI = hepatosomatic index, T = testosterone, E2 = estradiol-17β, 11-kT = 11-ketotestosterone.

Dietary Basic Ingredients(% crude protein)

Fish Size(g)

Feed Coefficient NPU(%)

References

Fish Meal (36.8) 5–8 1.2 ± 0.3 34.3 ± 8.2 Eckman (1987)Casein-Gelatin (47.3) 11.5 ± 1.6 1.2 ± 0.2 12.0 ± 2.0 Vasquez-Torres et al. (2002)Fish Meal (32.6) 11.5 ± 1.6 1.25 ± 0.3 32.0 ± 5.0Casein-Gelatin (56.6) 2.0 ± 0.03 0.77 ± 0.1 26.9 ± 1.7 Palacios et al. (2003)

Table 13. Progress in diet formulations for Piaractus and Colossoma spp. NPU = net protein utilization.


sides one P. fasciatum that was spermiating, no signs of sexual maturity were observed. Therefore, no attempt to induce final maturation was performed. In April 2002, thirteen fish (twelve P. fasciatum and one P. tigrinum) were captured in Pond 11 at IIAP and at Pucallpa. Water temperature ranged from 31.4 to 32.2oC, whereas dissolved oxygen ranged from 6.05 to 7.5 mg l-1. The reproductive characteristics of these fish are pre-sented in Tables 10 and 11.

Reproductive characteristics of the surubim raised in Colum-bus are summarized in Table 12. Histological analysis of the gonads was used to determine the sex of the fish. In females, ovaries were filled with oocytes at the perinucleolar stage, whereas lobules containing spermatogonia were observed in the testis of the males. Mean GSI and HSI reached 0.4 ± 0.2 and 0.6 ± 0.1% in females and 0.2 ± 0.1 and 0.7 ± 0.1% in males, respectively. Mean plasma testosterone was 208 ± 92 and 234 ± 57 pg ml-1 in females and males, respectively. Plasma estradiol-17α levels were low in females and were similar to those observed in males. 11-ketotestosterone levels were sig-nificantly higher in males (P < 0.05).

Discussion

Our results under Objective 1 suggest that dietary supplemen-tation of maca meal can increase growth rate and feed utiliza-tion in paco and could potentially improve utilization of other plant ingredients in order to replace fish meal. Previous studies conducted in the Aquaculture Laboratory (Lee and Dabrowski, in preparation) with rainbow trout alevins also showed growth-enhancing results in the maca meal-containing dietary group. This is a very important finding for the Peruvian aqua-culture industry considering that over 50% of production cost in fish farming is spent for feeds due to the soaring costs of fish meal proteins. Eventually, this will also open new markets for the Peruvian maca meal industry. The positive effects of maca meal need to be explored further in other frugivorous fishes of the Amazon rainforest. Table 13 summarizes our laboratory’s progress in developing “reference diets” for Piaractus and Colossoma spp. for further nutritional studies on these fish.

Our results under Objective 2 suggest the potential for using plasma sex steroid hormone analysis as a tool for quantita-tively assessing reproductive readiness of Pseudoplatystoma spp. broodstock. Further studies are needed to determine the plasma sex hormone levels of ovulating fish and post-spawned fish.

CONCLUSIONS

Effective larval rearing techniques for paco were developed that allow complete weaning to formulated diets. We anticipate that the automatic feeder system that we installed in our labo-ratory could be duplicated on a large-scale in IIAP hatcheries, especially Iquitos and Pucallpa Stations to ensure high larval survival and eventually artificial propagation of Amazonian fishes under semi-intensive conditions. Plasma sex steroid hormones have good potential for quantitatively assessing spawning readiness of broodstock for induced reproduction.

ANTICIPATED BENEFITS

This study aimed to investigate key aspects of the nutrition

and reproduction biology of several Amazonian freshwater fish species such as C. macropomum, Piaractus brachypomus, Pseudoplatystoma fasciatum, and P. tigrinum in order to improve or develop sustainable aquaculture technology for these spe-cies.

Through our collaborative effort with Peruvian investigators from IIAP-Iquitos and Pucallpa, we were able to develop the procedures of first-feeding of C. macropomum and Piaractus brachypomus and to monitor and understand the dynamics of gonadal steroidogenesis during maturation of Pseudoplatystoma 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 four-to-six week-old juveniles will dra-matically increase their survival and efficiency of production.

Catfish could be cultured in monoculture, or polyculture sys-tems in order to control the native cichlids like Cichlassoma and Aequidens. Both species reach large sizes (20 to 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. Pseudoplatystoma 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.

The graduate student who worked on this project, Maria Esther Palacios from the Universidad Nacional Mayor de San Marcos, has learned new techniques of weaning paco larvae to formulated feeds and had first-hand experience with the use of the automatic feeder system (Charlon and Bergot, 1992).

Collaborative studies between OhSU and Sao Paolo State University, Jaboticabal, Brazil, have been initiated to evaluate the diets and feeding systems developed at OhSU for weaning South American tropical fishes on a large scale.

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