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Diversification of Aquacultural Practices by Incorporation of Native Species and Implementation of Alternative Sex Inversion Techniques 10ATR3

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Diversification of Aquacultural Practices by Incorporation of Native Species and Implementation of Alternative Sex Inversion Techniques

Appropriate Technology Research 3 (10ATR3)/Study and Experiments/Mexico

Collaborating Institution
Universidad Juárez Autónoma de Tabasco, Mexico
      Wilfrido M. Contreras Sánchez
     Gabriel Márquez Couturier

Oregon State University
     Guillermo R. Giannico
     Carl B. Schreck

Objectives
1) Establish broodstock lots of tropical gar (Atractosteus tropicus) and mojarra castarrica (Cichlasoma urophthalmus).

2) Produce 100% male castarrica fry.

Significance
In the tropics, two approaches for utilizing the large diversity of fish species offer potential for aquaculture development: 1) species with moderate growth rates and excellent markets and 2) species with sexual dimorphism and large demand for the "attractively colored gender" in aquarium stores. In Southeastern Mexico, a growing number of fish producers are requesting the development of alternative culture techniques that involve native species. Since the early 1970s, the only species that has been available is the Nile tilapia and little effort has been devoted to develop options with local species. Worldwide, the administration of natural and synthetic steroids during early development of fish has been successfully used to induce sex inversion in several species (see reviews by Schreck, 1974; Hunter and Donaldson, 1983), and has become a common practice in the production of single sex populations to enhance productivity in the aquaculture industry. The protocols for the masculinization of 47 species and the feminization of 31 species have been developed so far (Pandian and Sheela, 1995). Among the steroids used, 17a-methyltestosterone (MT), trenbolone acetate (TA) and estradiol (E2) have proven effective for masculinizing (MT and TA) and feminizing (E2) fish fry.

At the Laboratory of Aquaculture in Universidad Juárez Autónoma de Tabasco (UJAT), the production of tropical gar fry has been successful over the last five years (Márquez-Couturier, 2000). A previous study demonstrated that females reach larger sizes than males (Contreras-Sánchez and Alemán-Ramos, 1987), but little is known regarding the advantages of producing 100% female populations. The tropical gar is considered a delicacy dish in Southern Mexico and an integral part of local culture—analogous to the importance of the salmon in the Northwest of the US. Gar is sold in every seafood restaurant in Tabasco and the pressure on its populations has lead to the listing of the species as "susceptible" (Contreras-Sánchez, 1990).

The mojarra castarrica has been considered as one of the few native species that may perform as well as tilapias in aquacultural systems in terms of growth rate and achievable harvest size (Martinez-Palacios and Ross, 1994). As in tilapias, males grow faster and larger than females; however, little has been done about the production of 100% male populations and the few studies that have been carried out resulted in less than 70% male populations (Hernández-Betancourt, 1988).

The growing international demand and high prices for tropical species in aquarium stores has already created great pressure on the natural populations. As an example, in Japan, tropical gar juveniles have a wholesale price of US$15 per fish. Many of the specimens sold in aquaria are wild-captures and very few are produced in aquacultural facilities. In the last two years, the laboratory of aquaculture has started research on native species that show potential for being incorporated into intensive culture systems. We are currently studying the early life history of tropical gar, mojarra castarrica, mojarra paleta and mojarra azuleja, and we are working on a manual for the culture of these species. These on-going studies involve the time required for embryonic and larval stages, time of sex differentiation, feeding habits, etc.

Possible alternatives to the problems encountered for sex inversion of new species of fish in aquaculture are the administration of steroids via bioencapsulation and immersions. The first technique offers the advantage that the larvae of the two species proposed for this study strongly prefer live food over artificial diets. This technique has been used for administering chemotherapeutics and nutrients to fish, shrimps and prawns (Roque et al., 1998; Touraki et al., 1999) and is considered as a good alternative method to incorporation of hormone into manufactured feeds. The second alternative offers the possibility of feminizing or masculinizing the proposed species without the need of feed containing hormones. Because masculinization of fry by oral administration of MT is considered the most successful method employed to masculinize fish fry (Green et al., 1997), we intend to compare this protocol with the administration of bioencapsulated trenbolone acetate (TA) and immersions in TA. We will also validate the bioencapsulation technique using tilapia as a model species. For feminization of tropical gar, immersions in estradiol (E2) will be evaluated. We will use the same protocol that we have used to masculinize Nile tilapia (i.e., 3­5 h immersions at the onset of feeding; Contreras, 2001) after determining the time at which the gonads enter the labile period (work which is currently underway and expected to yield results within 6 months).

Extension workshops and outreach material will be developed to increase the awareness of both producers and the public about the economic potential and the environmental benefits associated with the diversification of aquaculture by incorporating native species of fish.

Anticipated Benefits
Incorporation of native species to aquaculture is needed. The development of technological packages for native species will benefit fish farmers in Southern Mexico and Central America. Particularly, this study will focus on the development of techniques that allow sex inversion in those species of economic importance. These techniques are of considerable importance in promoting diversification of aquaculture species. Furthermore, the assessment of techniques that do not require the use of steroid-treated food may provide aquaculturists with a safe and cost effective alternative, because immersion will require substantially shorter exposure periods and the steroid will be contained for controlled filtration or biodegradation. If the proposed protocols provide positive results, farm trials will be set up at Ejido Rio Playa and workshops will be conducted to train farmers, students and technicians.

Research Design
Study A: Enrichment and Detection of E2 and TA in Artemia Nauplii

Site: Experiments will be conducted at UJAT, and samples processed at Oregon State University, Corvallis, OR.

Methods: Immersion of Artemia nauplii in steroids (E2 or TA) 1,000 µg/l. Two methods for Artemia enrichment will be evaluated (bioencapsulation via oil diets and immersion in steroid solutions).

Laboratory and Pond Facility: UJAT; 20 4-l glass jars. Oregon State University; Laboratory and equipment needed for RIA and HPLC.

Culture Period: 12 hrs

Stocking Density: 1 million nauplii/L

Test Species: Artemia salina (and/or Daphnia magna depending upon Artemia availability)

Nutrient Inputs: None

Water Management: None

Sampling schedule: The experiment consists of three treatments:

Each treatment will consist of three replicates. Water with Artemia nauplii (50 ml) samples will be collected from the glass jars at 0, 2, 4, 6, and 12 hours. Nauplii will be washed in nanopure water, dried and samples will be frozen (­20°C) and preserved until processing. All samples will be extracted using ether and the concentration of steroid determined by RIA (E2) or HPLC (TA).

Statistical Methods and Hypothesis: H01: E2 and TA are not detectable in nauplii at any sampling time. This part of the study is descriptive and therefore, statistical analysis is unnecessary for testing the null hypothesis (i.e., detection of any amount of E2 or TA in nauplii will be sufficient for rejecting the null hypothesis).

Schedule: Data collection, 7/01-8/01; Technical report, 6/30/02

Experiment B: Validation of Bioencapsulation Masculinization of Nile Tilapia Fry

Site: Experiments will be conducted at Oregon State University, Corvallis, OR.

Methods: Oral administration of TA via bioencapsulation using Artemia nauplii in 200-l fiberglass tanks.

Laboratory and Pond Facility Fish Performance and Genetics Laboratory, OSU. 2 recirculating systems with 12 200-l tanks each. 15 females and 5 males for production of fry.

Culture Period: 4 months or until tilapias reach sampling size.

Stocking Density: 100 fry/tank

Test Species: Nile Tilapia (Oreochromis niloticus).

Nutrient Inputs: None

Water Management: Water will be maintained at 28°C, 25% water exchange once a week.

Sampling Schedule: The experiment consists of four treatments:

Each treatment will be triplicated. At the end of 4-month grow-out period, a subsample of the tilapias in each experimental unit (50) will be killed with an overdose of anesthetic (MS-222) to determine if the treatment with TA resulted in masculinization. The following water quality parameters will be measured daily: pH, DO, and temperature.

Statistical Methods and Hypothesis: H01: Administration of TA to tilapia fry results in treatments with the same sex ratios as those of the controls. The efficacy of TA treatment will be tested comparing between treatments by Chi-squared test.

Schedule: Data collection, 9/01-7/02; Technical report, 3/30/03

Experiment C: Feminization of Tropical Gar Fry

Site: Experiments will be conducted at the Laboratory of Aquaculture at UJAT, Tabasco, México.

Methods: Oral administration of E2 via bioencapsulation using Artemia nauplii in fiberglass tanks (1 m3); and immersion in E2

Laboratory and Pond Facility: Universidad Juárez Autónoma de Tabasco; 20 50-l aquaria, 3 reproduction concrete tanks (8 m2), 50 net cages (1 m3) for grow-out, 3 grow-out ponds (200 m3), a total of 10 females and 30 males for production of fry.

Culture Period: 6 months (gars) or until fish reach sampling size

Stocking rate: 50 fry/tank

Test Species: Tropical gar (Atractosteus tropicus)

Nutrient Inputs: None

Water Management: 50% water exchange will be performed twice a week.

Sampling schedule: For each species, the experiment will consist of seven treatments:

Each treatment will be triplicated. At the end of 6 month grow-out period, gars will be killed with an overdose of anesthetic (MS-222) to determine if the treatment with E2 resulted in feminization. The following water quality parameters will be measured daily: pH, DO, and temperature.

Statistical Methods and Hypothesis: H01: Administration of E2 to gar fry results in treatments with the same sex ratios as those obtained in the control treatment. The efficacy of E2 treatment will be tested comparing between treatments by Chi-squared test.

Schedule: Data collection, 9/01-5/02; Technical report, 6/30/02

Experiment D: Masculinization of Castarrica Fry

Site: Experiments will be conducted at the Laboratory of Aquaculture at UJAT, Tabasco, México.

Methods: Oral administration of TA via bioencapsulation using Artemia nauplii in fiberglass tanks (1 m3) and immersion in TA.

Laboratory and Pond Facility: Universidad Juárez Autónoma de Tabasco; 20 50-l aquaria, 3 reproduction concrete tanks (8 m2), 50 net cages (1 m3) for grow-out, 3 grow-out ponds (200 m3).

Culture Period: 4 months or until castarricas reach sampling size.

Stocking Density: 50 fry/tank

Test Species: Mojarra castarrica (Cichlasoma urophthalmus).

Nutrient Inputs: None

Water Management: 50% water exchange will be performed twice a week.

Sampling Schedule: The experiment consists of six treatments:

Each treatment will be triplicated. At the end of 4-month grow-out period, castarricas will be killed with an overdose of anesthetic (MS-222) to determine if the treatment with TA resulted in masculinization. The following water quality parameters will be measured daily: pH, DO, and temperature.

Statistical Methods and Hypothesis: H01: Administration of TA to castarrica fry results in treatments with the same sex ratios as those obtained in the controls. The efficacy of TA treatment will be tested comparing between treatments by Chi-squared test.

Schedule: Data collection, 7/02-2/03; Technical report, 3/30/03

Literature Cited
Contreras-Sánchez, W., 1990. Monitoreo de las poblaciones de pejelagarto Atractosteus tropicus en el estado de Tabasco, México. Informe técnico. SEDUE, 60 pp.

Contreras-Sánchez, W., 2001. Sex Determination in Nile Tilapia, Oreochromis niloticus: Gene Expression, Masculinization Methods, and Environmental Effects Ph.D. thesis, Oregon State University, Corvallis, Oregon.

Contreras-Sánchez, W. and L. Alemán-Ramos, 1987. Aspectos reproductivos y desarrollo embrionario del pejelagarto Lepisosteus tropicus (Gill), en el estado de Tabasco. Memorias del IX congreso nacional de Zoología. 13­16 de Octubre. Tabasco, México.

Green, B.W., K.L. Veverica, and M.S. Fitzpatrick, 1997. Fry and fingerling production. In: H. Egna and C. Boyd (Editors), Dynamics of Pond Aquaculture. CRC Press, Boca Raton, Florida, pp. 215­243.

Hernández-Betancourt, S., 1988. Inversión sexual de la mojarra Cichlasoma urophthalmus a través de la aplicación de la 17-a-metiltestosterona. Masters degree thesis, CINVESTAV-IPN-Unidad Mérida, México, 66 pp.

Hunter, G.A.. and E.M. Donaldson, 1983. Hormonal sex control and its application to fish culture. In: W.S. Hoar, D.J. Randall, and E.M. Donaldson (Editors), Fish Physiology. Academic Press, New York, pp. 223­303.

Martínez-Palacios and L. Ross, 1994. Biología y cultivo de la mojarra latinoamericana Cichlasoma urophthalmus. CONACYT, México, 203 pp.

Pandian, T.J., and S.G. Sheela, 1995. Hormonal induction of sex reversal in fish. Aquaculture, 138:1­22.

Roque, A., J.F. Turnbull, and B. Gomez-Gil, 1998. Delivery of bioencapsulated oxytetracycline to the marine shrimp Penaeus monodon. J. World Aquacult. Soc., 29:249­251.

Schreck, C.B., 1974. Hormonal treatment and sex manipulation in fishes. In C.B. Schreck (Editor), Control of Sex in Fishes. Virginia Polytechnic Institute and State University Extension Division, Blacksburg, Virginia, pp. 84­106.

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