Reproduction Control Research 2

Note: Experimental Design for 8RCR2A has been revised. See First Addendum to the Eighth Work Plan
Note: Experimental Design for 8RCR2B has been revised. See First Addendum to the Eighth Work Plan

Collaborating Institutions
Objective
Significance
Anticipated Benefits
Collaborative Arrangements
Experimental Design
Investigations

Immersion of Tilapia Fry in MDHT
Effect of Fish Density on Efficacy of Masculinization by Immersion in MDHT
Masculinization of Tilapia Fry by Immersion in MDHT at a Production Level

Collaborating Institutions

Oregon State University

Martin Fitzpatrick

Carl Schreck

Auburn University

Ronald Phelps

Objective

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

Significance

The experiments within this objective will address several constraints to aquacultural productivity, namely the limited knowledge of reproductive physiology and limited use of new hatchery technologies for improved strategies of fingerling production. 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

not applicable

Experimental Design

Previous experiments have demonstrated that tilapia fry can be masculinized by immersion in 17a-methyldihydrotestosterone (MDHT) for 3 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 on age of fry is unavailable).

Immersion of Tilapia Fry in MDHT

(Reproduction Control Research 2A)

Note: Experimental Design for 8RCR2A has been revised. See First Addendum to the Eighth Work Plan

Site: Oregon State University, Corvallis, OR

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

Other Inputs: none

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

Sampling Plan: The experiment consists of 6 groups: 1) fry immersed in MDHT at 500 µg/L for 3 hr on 10 and 13 days post-fertilization (dpf); 2) fry immersed in MDHT at 500 µg/L for 3 hr on 10 dpf; 3) fry immersed in MDHT at 500 µg/L for 3 hr on 13 dpf; 4) fry fed MT at 60 mg/kg food for 28 days; and 5) fry immersed in water that contains the same volume of ethanol used to dissolve the MDHT; 6) fry not immersed and fed regular feed. Each group will be replicated. 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 MT resulted in masculinization.

Statistical Methods and Hypotheses: H₀1: Immersion of tilapia in MDHT results in groups with the same sex ratio as control fish; H₀2: Immersion of tilapia for one 3-hr exposure on 10 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, 10/96-4/97; technical report, 6/97.

Note: Experimental Design for 8RCR2B has been revised. See First Addendum to the Eighth Work Plan

Site: Oregon State University, Corvallis, OR

Laboratory Facilities: Oregon State Universitytwo aquaria containing a total of two males and six females for production of fry, 3 L chambers for immersion treatment, and 40 L 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^oC.

Other Inputs: none

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

Sampling Plan: The experiment consists of fry immersed in MDHT at 500 µg/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 densities:

1) 33 fish/L

2) 66 fish/L

3) 100 fish/L

4) 200 fish/L

5) 300 fish/L

In addition, a control group will be included consisting of unimmersed fry held at 33 fish/L during immersion of the other groups. All groups will be returned to 8 fish/L between immersions and during grow-out. All groups will be replicated.

Statistical Methods and Hypotheses: H₀1: Immersion in MDHT of tilapia held at different densities results in groups with the same sex ratio as control fish; H₀2: 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.

Site: Auburn University, Auburn, AL

Pond Facilities: Auburn Universityone pond for broodstock and one pond for fry grow-out; aquaria for treatment of fry.

Culture Period: 21 days for broodstock; 180 days for offspring.

Stocking Rate: brood stock: 200 females and 100 males; fry for sex reversal: 5,000 fry/m² (equivalent to 8 fry/liter in 60 cm deep hapa).

Water Management:

Other Inputs: none

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

Sampling Plan: Fry will be collected 17 days after stocking the brood pond with adults. The fry will be graded for size by passing them through fine mesh such that only fish between 8 and 12 mm length will be used in the experiment. The exact nature of the treatments depends on the outcome of Study 1; i.e. if Study 1 demonstrates that two immersions are necessary for masculinization, then the study will focus on determining if immersions on any two days between collection of the fry (Day 1) and Day 4 results in masculinization. The treatment groups will be:

1) fry immersed in MDHT at 500 µg/L for 3 hr on Day 1 and Day 4;

2) fry immersed in MDHT at 500 µg/L for 3 hr on Day 1 and Day 2;

3) fry immersed in MDHT at 500 µg/L for 3 hr on Day 3 and Day 4;

4) fry fed MT at 60 mg/kg food for 28 days;

5) fry not immersed and fed regular feed.

If Study 1 demonstrates that a single immersion is effective for masculinization, then the study will focus on determining if immersion on any of the first four days after collecting fry results in masculinization. The treatment groups will be:

1) fry immersed in MDHT at 500 µg/L for 3 hr on Day 1;

2) fry immersed in MDHT at 500 µg/L for 3 hr on Day 2;

3) fry immersed in MDHT at 500 µg/L for 3 hr on Day 3;

4) fry immersed in MDHT at 500 µg/L for 3 hr on Day 4;

5) fry fed MT at 60 mg/kg food for 28 days;

6) fry not immersed and fed regular feed.

Each group will be replicated. 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 MT resulted in masculinization.

Statistical Methods and Hypotheses: H₀1: Immersion of tilapia in MDHT results in groups with the same sex ratio as control fish; H₀2: Immersion of tilapia on different days results in the same sex ratio as all treatment groups. Sex ratios will be compared by Chi-squared test.

Deliverables: A technical report and potential journal manuscript.

Schedule: Data collection, 4/97-2/98; technical report, 3/98.

In Year 3 (and 4), experiments similar to Study 3 will be conducted in Honduras and Thailand to investigate applicability of immersion techniques between CRSP sites.