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Research Projects - PD/A CRSP 18th Admin Report
PD/A CRSP Eighteenth Annual Administrative Report

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Research Projects
Mexico Project

MOU No. RD009C

Staff

Oregon State University, Corvallis, Oregon
Martin S. Fitzpatrick US Co-Principal Investigator, US Regional Coordinator (through April 2000)
Carl B. Schreck US Co-Principal Investigator
Wilfrido M. Contreras-Sánchez Graduate Research Assistant (Mexico; CRSP funded)
John Hayes Graduate Research Assistant (Uruguay/USA; from July 2000; CRSP funded)
Carisska Anthony Undergraduate Student Worker (USA; from April 2000)
Kelly Callison Undergraduate Student Worker (USA; through September 1999)
Jesse Chastain Undergraduate Student Worker (USA; from October 1999)
Janine Gonzalez Undergraduate Student Worker (USA; from August 1999)
Nancy Savage Undergraduate Student Worker (USA; through January 2000)

Universidad Juárez Autónoma de Tabasco, Villahermosa, Mexico
Gabriel Márquez-Couturier Host Country Co-Principal Investigator
Wilfrido M. Contreras-Sánchez Host Country Co-Principal Investigator
Mario Fernandez Professor
Ulises Hernandez Vidal Technician
Alejandro MacDonald VeraTechnician
Guadalupe Morales Lara Technician
Luis Arturo Dorantes Lopez Undergraduate Student
Sofia Carolina Santiago Ruiz Undergraduate Student

Site Background

The PD/A CRSP has been active in Mexico since 1997. A Memorandum of Understanding (MOU) was signed between Oregon State University (OSU) and the Universidad Juárez Autónoma de Tabasco (UJAT) in June 1999. Until now, research conducted in Mexico has been reported as either Reproduction Control Research or Effluents and Pollution Research. In 2000, due to exclusive collaboration between OSU and UJAT, research conducted under the differing research themes was consolidated to form the Mexico Project. CRSP research in Mexico emphasizes reproduction control through the use of hormone immersion and examines the fate of masculinizing agents in effluents.

Work Plan Research

The following Ninth Work Plan investigations continued into the current reporting period:

This MOU was also awarded funding to conduct the following Ninth Work Plan investigations:

Note: The schedule for 9RCR5C has been modified. The methods and schedules for 9RCR5B and 9ER2B have been modified. Please see Appendix 5, "Completion Dates for Work Plan Studies," for revised schedule information. The revised methods will appear in the Addendum to the Ninth Work Plan.

Networking

Throughout the course of this year, OSU researchers have been in contact with Amrit Bart, CRSP researcher on the Thailand Project, regarding collaboration on an investigation of masculinization of tilapia. OSU researchers sent information to Bart regarding the purchase of steroids in the US and Thailand, as well as their experimental design for masculinizing Nile tilapia by immersion in trenbolone acetate. A graduate student will be using this technique in Bart's laboratory.

After presenting two papers at the V Central American Symposium on Aquaculture and the Aquaculture America 2000 meeting, Wilfrido Contreras-Sánchez received several requests for the group's published information regarding tilapia masculinization protocols with the use of steroids and information for determining the fate of MT in the pond environment. The researchers sent descriptions of their methods and results to scientists and farmers in Mexico, Costa Rica, Honduras, Nicaragua, Brazil, Ecuador, Venezuela, Thailand, and the US.

Contreras shared information on PD/A CRSP masculinization techniques with Javier Romero, a student at the Universidad Tecnica de Machala, Ecuador. Contreras reviewed Romero's bachelor's degree thesis, entitled "Evaluation of bio-enriched Daphnia sp. for sex inversion of red tilapia fry."

Contreras is a participant in an online discussion group on tilapia culture in which he interacts with tilapia growers from sites around the world. As a result of these conversations, the researchers have sent copies of the group's methods and published results to growers in Mexico, Brazil, Ecuador, Venezuela, Thailand, and the US.

Martin Fitzpatrick and Contreras made an agreement with two professors from the Universidad Autónoma Metropolitana and the Instituto Politecnico Nacional, both in Mexico City, regarding a research project that was submitted to the National Council for Science and Technology (Mexico) for funding. If approved, the researchers will collaborate on the construction of experimental design; training of students, technicians, and researchers on safe handling of steroids; sex inversion by immersion trials; sex identification using histological methods; and data analysis.

Gabriel Márquez-Couturier has initiated collaborative research with Mario Fernández from the aquaculture school at UJAT. Their proposed studies involve masculinization of Nile tilapia fry through large-scale immersions and additional comparison trials to MT-fed groups. Márquez-Couturier additionally trained 12 students and a professor on the safe handling of synthetic steroids and masculinizing techniques. He has also been working closely with farmers from Rio Playa in Tabasco on a project involving the use of native species (Atractosteus tropicus, Cichlasoma urophthalmus, and Petenia splendida) and Nile tilapia in aquaculture. Márquez-Couturier has also started a number of small projects involving tilapia culture in hapas and earthen ponds using sex-inverted tilapia from CRSP project technology.

CRSP researchers at OSU provided juvenile Nile tilapia to a beginning aquaculturist who is experimenting with the feasibility of hydroponics using water from a tilapia system as the primary nutrient source. The aquaculturist plans to grow basil plants in his recirculating system and has obtained the necessary permits to grow and distribute tilapia. He hopes to become a tilapia fry distributor for Oregon.

Educational Outreach

CRSP researchers at OSU provided tilapia fry to Mike Tyler, a teacher at Arcadia Elementary School in Toledo, Oregon. Tyler's students grew the tilapia to learn about aquaculture, and they sold the adult fish in the local market when they were finished. The researchers also provided fry to Doug Ernst, PD/A CRSP Database Manager and Research Associate on Decision Support Systems Research, for use at Corvallis High School.

Fitzpatrick used examples from his PD/A CRSP–sponsored research in an OSU Fish Physiology course.

Contreras advised students in OSUs Department of Fisheries and Wildlife who were taking a problem-solving class. The students proposed and developed a project to evaluate the growth of Nile tilapia juveniles under different density conditions. Contreras provided them information on the principles for setting experimental designs, tilapia culture, and data analysis, and he provided juvenile tilapia for use in the project.

Publications

Contreras-Sánchez, W., M.S. Fitzpatrick, G. Márquez-Couturier, and C.B. Schreck, 1999. Masculinization of the Nile tilapia (Oreochromis niloticus) by immersion in synthetic androgens: Timing and efficacy. In: B.W. Green, H.C. Clifford, M. McNamara, and G.M. Montaño (Editors), V Central American Symposium on Aquaculture. San Pedro Sula, Honduras, pp. 246–248.
Contreras-Sánchez, W.S., M. Fitzpatrick, R.H. Milston, and C.B. Schreck, 2000. Masculinization of Nile tilapia: Alternate treatments and environmental effects. In: B. Norberg, O.S. Kjesbu, G.L. Taranger, E. Andersson, and S.O. Stefansson (Editors), Proceedings of the Sixth International Symposium on the Reproductive Physiology of Fish. Institute of Marine Research and University of Bergen, Bergen, Norway, pp. 250–252.
Fitzpatrick, M., W. Contreras-Sánchez, R.H. Milston, and C.B. Schreck, 1999. Fate of masculinizing agent methyltestosterone in the pond environment. In: B.W. Green, H.C. Clifford, M. McNamara, and G.M. Montaño (Editors), V Central American Symposium on Aquaculture. San Pedro Sula, Honduras, pp. 249–250.
Gale, W.L., M. Fitzpatrick, M. Lucero, W.M. Contreras-Sánchez, and C.B. Schreck, 1999. Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in androgens. Aquaculture, 178(1999):349–357.

Presentations

Contreras-Sánchez, W.M., M.S. Fitzpatrick, and C.B. Schreck. Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in trenbolone acetate. Presented to Aquaculture America 2000 at New Orleans, Louisiana, 2–5 February 2000.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, R.H. Milston, and C.B. Schreck. Masculinization of Nile tilapia with steroids: Alternate treatments and environmental effects. Presented to the Gilbert Ichthyological Society Meeting at Newport, Oregon, 18 October 1999.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, M. Alonso, C.B. Schreck, and J.C. Leong. Identification of unique genes expressed during sex inversion of Nile tilapia (Oreochromis niloticus) induced by short immersions in the synthetic steroid trenbolone acetate. Presented to Fourth International Symposium on Fish Endocrinology at Seattle, Washington, 31 July–3 August 2000.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, M. Alonso, C.B. Schreck, and J.C. Leong. Identification of unique genes induced by trenbolone acetate during sex inversion of Nile tilapia (Oreochromis niloticus). Presented to the Eleventh Western Regional Conference on Comparative Endocrinology at Corvallis, Oregon, 24–25 March 2000.
Fitzpatrick, M.S., W.M. Contreras-Sánchez, and C.B. Schreck. Methyltestosterone persists in the environment after use for masculinizing Nile tilapia. Presented to Aquaculture America 2000 at New Orleans, Louisiana, 2–5 February 2000.

Conferences

V Central American Symposium on Aquaculture at San Pedro Sula, Honduras, 18–20 August 1999. (Contreras, Márquez-Couturier)
The Gilbert Ichthyological Society Meeting at Newport, Oregon, 18 October 1999. (Contreras).
PD/A CRSP Annual Meeting at New Orleans, Louisiana, 31 January–2 February 2000. (Fitzpatrick, Contreras, Márquez-Couturier)
Aquaculture America 2000 at New Orleans, Louisiana, 2–5 February 2000. (Fitzpatrick, Contreras, Márquez-Couturier)
Eleventh Western Regional Conference on Comparative Endocrinology at Corvallis, Oregon, 24–25 March 2000. (Fitzpatrick, Contreras)
Fourth International Symposium on Fish Endocrinology at Seattle, Washington, 31 July–3 August 2000. (Contreras)

Award

Wilfrido Contreras-Sánchez was awarded the Savery Outstanding Graduate Student Award, given by the College of Agricultural Sciences, Oregon State University, at a ceremony on 18 May 2000.

Masculinization of Tilapia by Immersion in Trenbolone Acetate: Growth Performance of Trenbolone Acetate–Immersed Tilapia

Ninth Work Plan, Reproduction Control Research 5B (9RCR5B)
Final Report

Wilfrido M. Contreras-Sánchez and Martin S. Fitzpatrick
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Carl B. Schreck
Oregon Cooperative Fishery Research Unit
Biological Resources Division—US Geological Survey
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Abstract

Preliminary studies in our laboratory showed that the synthetic androgen trenbolone acetate (TA) is a good candidate for masculinizing Nile tilapia (Oreochromis niloticus) fry using short immersions. In this study, we investigated the effects of TA treatment on the growth performance of Nile tilapia. We tested the potential anabolic effects of two treatments by growing treated and control fish for 81 and 114 days. Our results suggest that masculinizing treatments involving short-term immersions in TA and 4-week feeding with 17alpha-methyltestosterone (MT) do not result in significant increases in fish growth. Despite significant masculinization (65 to 70% with TA and 100% with MT) in both treatments, we found no differences in final weight between treatments.

Masculinization of Tilapia by Immersion in Trenbolone Acetate: Detection of Trenbolone Acetate after Treatment

Ninth Work Plan, Reproduction Control Research 5C (9RCR5C)
Progress Report

Wilfrido M. Contreras-Sánchez and Martin S. Fitzpatrick
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Carl B. Schreck
Oregon Cooperative Fishery Research Unit
Biological Resources Division—US Geological Survey
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Abstract

In previous experiments we have found that two 3-hour immersions in trenbolone acetate (TA) can successfully masculinize Nile tilapia fry. In this study we are investigating how the concentration of TA in the immersion water changes before and after treatment to determine the amount of hormone uptake and estimate the potential for reuse of the treatment water. Nile tilapia fry were subjected to two 3-hour immersions at 11 and 13 days post-fertilization (dpf) in water containing 500 ml of TA. Surprisingly, we have found that the concentration of TA before and after treatment is highly variable and below the expected levels. We are currently assessing whether TA comes out of solution and forms precipitates or binds to the jar glass.

Fate of Methyltestosterone in the Pond Environment: Detection of MT in Pond Soil from a CRSP Site

Ninth Work Plan, Effluents and Pollution Research (9ER2B)
Final Report

Wilfrido M. Contreras-Sánchez and Martin S. Fitzpatrick
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Carl B. Schreck
Oregon Cooperative Fishery Research Unit
Biological Resources Division—US Geological Survey
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Abstract

The following study examined the persistence of 17alpha-methyltestosterone (MT) in the environment after its use for masculinizing Nile tilapia in nursery ponds located at two CRSP sites: Sagana Fish Farm, Kenya, and the Universidad Juárez Autónoma de Tabasco (UJAT), Mexico. Fry were treated with a masculinizing dose of MT (60 mg kg-1) for four weeks beginning at the initiation of feeding. Concentrations of MT were determined by radioimmunoassay, which revealed that the levels of MT in the sediments from the Sagana Fish Farm had a mean value of 4,567 pg g-1. The concentration of MT slightly decreased near the drain of the pond. Concentration of MT in water and sediments from UJAT showed background levels after first-time use of MT in the pond. These results suggest that accumulation of MT may take place after masculinization of a significant number of fish.

Fate of Methyltestosterone in the Pond Environment: Impact of MT-Contaminated Soil on Tilapia Sex Differentiation

Ninth Work Plan, Effluents and Pollution Research 2C (9ER2C)
Final Report

Wilfrido M. Contreras-Sánchez and Martin S. Fitzpatrick
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Carl B. Schreck
Oregon Cooperative Fishery Research Unit
Biological Resources Division—US Geological Survey
Department of Fisheries and Wildlife
Oregon State University
Corvallis, Oregon, USA

Abstract

The following study examined the effect of environmentally persistent 17alpha-methyltestosterone (MT) on sex differentiation of Nile tilapia (Oreochromis niloticus). Three different broods of fry were treated one after the other with a masculinizing dose of MT (60 mg kg-1) for four weeks beginning at the initiation of feeding in model ponds which consisted of 60-l tanks that contained 5 kg of soil. Four weeks after the last MT treatment, two different broods of tilapia fry were fed control feed while being maintained in the tanks that had contained the MT-treatment groups. Water and soil samples were taken before the onset of treatment and on the last day of treatment during each treatment cycle. Concentrations of MT were determined by radioimmunoassay, which showed that the levels of MT in the water were elevated between about 200 and 1,250 pg ml-1 during the three cycles of MT, and then returned to background levels during the remaining cycles of control diet feeding. Analysis of water samples taken shortly after adding the MT-impregnated food to the tanks revealed that MT leaks into the water within a minute of treatment. The levels of MT in the soil were elevated to about 2,000 pg g-1 after one feeding cycle and remained elevated between 1,400 and 3,300 pg g-1 through three months after the conclusion of the last MT feeding cycle, including the time during which the control-fed fry were raised in these tanks. The sex ratios of the groups fed control food while being maintained in the tanks that had contained the MT-treatment groups were not different from control fish; however, several individuals in the former groups had intersexual gonads, suggesting some impact on development.

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