REPRODUCTION CONTROL RESEARCH

Objective 1
To determine the optimal treatment conditions for masculinization of tilapia by immersion in 17a-methyldihydrotestosterone (mestanolone).

Significance
All-male populations of tilapia provide several important advantages for aquaculture, including enhanced growth (males grow faster and larger) and prevention of unwanted reproduction (which diverts energy away from somatic growth). Dietary treatment with 17a-methyltestosterone (MT) has been shown to be an effective means of producing all-male tilapia populations; however, the treatment requires a minimum of several weeks exposure. Development of techniques for masculinization through immersion in 17a-methyldihydrotestosterone (mestanolone)- containing solutions may provide aquaculturists with a safe and cost effective alternative to treating fry with food that contains MT, because immersion will require substantially shorter exposure periods and the steroid will be contained for controlled filtration or biodegradation.

Anticipated Benefits
Development of immersion in steroid as an alternative treatment for masculinizing tilapia will minimize treatment time and potentially increase efficiency of exposure and safety in handling masculinizing steroids.

Collaborative Arrangements
N/A

Experimental Design
Previous experiments have demonstrated that tilapia fry can be masculinized by immersion in 17a-methyldihydrotestosterone (MDHT) for three hours on Day 10 and on Day 13. During the first year, studies will be conducted to determine if a single immersion on either day is as effective for masculinization as the two day exposure or feeding MT. During the second year, studies will be conducted to determine if increasing densities of tilapia fry can be masculinized with the optimal immersion protocol (including timing and concentration) and if the optimal immersion protocol is effective when adapted to a production setting (when precise information of age of fry is unavailable).

Site: Oregon State University, Corvallis, Oregon

Laboratory Facilities: Oregon State University—two aquaria containing a total of two males and six females for production of fry, 3L chambers for immersion treatment , and 40L-aquaria for raising the fry.

Culture Period: 180 days for offspring.

Stocking Rate: 33 fry/liter for immersion treatment; 8 fish/liter for grow out.

Water Management: Water temperature will be maintained at 28-30&Mac251;C.

Other Inputs: None

Test Species: Nile tilapia (Oreochromis niloticus), Ivory Coast strain.

Sampling Plan: The experiment consists of 5 groups:

1) fry immersed in MDHT at 500mg/L for 3hr on 10 and 13 days post-fertilization (dpf);
2) fry immersed in MDHT at 500mg/L for 3hr on 10 dpf;
3) fry immersed in MDHT at 500mg/L for 3hr on 11 dpf;
4) fry immersed in MDHT at 500mg/L for 3hr on 13 dpf;
5) fry immersed in water that contains the same volume of ethanol used to dissolve the MDHT.

Each group will be triplicated. At the end of the 4 month grow out period, the tilapia will be killed and the gonads examined to determine if the treatment with MDHT resulted in masculinization.

Statistical Methods and Hypotheses: Ho 1: Immersion of tilapia in MDHT results in groups with the same sex ratio as control fish; Ho 2: Immersion of tilapia for one 3-hr exposure on 10, 11, or 13 dpf results in the same sex ratio as two 3-hr immersions on 10 and 13 dpf. Sex ratios will be compared by Chi-squared test.

Deliverables: A technical report and potential journal manuscript.

Schedule: Data collection, 12/96–6/97; technical report, 10/97.

Site: Oregon State Univesity, Corvallis, Oregon

Laboratory Facilities: Oregon State University—two aquaria containing a total of two males and six females for production of fry, 3L–chambers for immersion treatment, and 40L-aquaria for raising the fry.

Culture Period: 180 days for offspring.

Stocking Rate: variable during treatment; 8 fish/liter for grow out.

Water Management: Water temperature will be maintained at 28-30&Mac251;C.

Other Inputs: None

Test Species: Nile tilapia (Oreochromis niloticus ), Ivory Coast strain.

Sampling Plan: The experiment consists of fry immersed in MDHT at 500mg/L for 3 hr on 10 and 13 days post-fertilization (dpf) [exposure will be changed if Study 1 indicates that a single exposure sufficiently masculinizes tilapia] at the following densitites:

1) 33 fish/L
2) 66 fish/L
3) 100 fish/L
4) 200 fish/L

In addition, a control group will be included consisting of fry held at 33 fish/L during immersion in water containing ethanol. All groups will be returned to 8 fish/L between immersions and during grow-out. All groups will be triplicated.

Statistical Methods and Hypotheses: Ho 1: Immersion in MDHT of tilapia held at different densities results in groups with the same sex ratio as control fish; Ho 2: Immersion of tilapia held at different densities results in groups with the same sex ratios. Sex ratios will be compared by Chi-squared test.

Deliverables: A technical report and potential journal manuscript.

Schedule: Data collection, 6/97–4/98; technical report, 6/98.

Objective 2
To determine if 17a-methyltestosterone (MT) persists in the pond environment after dietary treatment of tilapia with MT.

Significance
The use of all-male populations of tilapia for culture offers several important advantages, including enhanced growth (males grow faster and larger) and prevention of unwanted reproduction ( which diverts energy away from somatic growth). Treatment with 17a-methyltestosterone (MT)- impregnated food has been shown to be an effective means of producing all-male tilapia populations. However, signifigant "leakage" of steroids such as MT into the pond environment may occur from uneaten or unmetabolized food and thus pose a risk of unintended exposure of hatchery workers as well as fish or other non-target organisms. Therefore, determining the fate of MT in semi-closed systems such as ponds will yield important information on both safety and efficacy of MT use for masculinization.

Anticipated Benefits
An assessement of MT persistence and fate in the water derived from dietary MT treatment will provide information on the potential risks of using this technology to workers and to non-targeted fish. Furthermore, determing when MT no longer can be detected in the pond environment will help to establish safety guidelines for the use of MT.

Collaborative Arrangements
None

Experimental Design
Analytical procedures for measuring MT and other potential masculinizing metabolites in the pond environment will be developed using High Performance Liquid Chromatography (HPLC). A radioimmunoassay (RIA) will be set up to measure the quantity of MT. During the first year, an experiment will be conducted using 3L-chambers as model systems; subsequent experiments will be conducted using production
ponds at Auburn and if possible in Honduras and Thailand.

Reproductive Control Research 2C remains unchanged.