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lanky
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just a little fun post
Objectives
1) To determine if a single immersion of tilapia fry in trenbolone acetate results in masculinization.
2) To determine the growth performance of trenbolone acetate-immersed tilapia.
3) To determine how long trenbolone acetate persists in the water and treatment container.
Significance
Masculinization of tilapia fry by dietary treatment with anabolic steroids such as 17a-methyltestosterone (MT) is common in aquaculture (Green et al., 1997); however, other methods for masculinization of tilapia such as immersion in steroid-containing solutions are not well developed. In CRSP experiments conducted for the Eighth Work Plan, MT feeding was shown to result in the persistence of MT in soil for at least several weeks after the end of treatment. Such MT in the pond environment may pose a health risk to workers and exposure risk to other organisms, including fish, in the pond environment.
The use of immersion technology has the advantage that the steroid solution in contained within the treatment vessel, and thus can be easily processed. In previous CRSP experiments, Gale et al. (1995) demonstrated that two 3-hr immersions of tilapia fry in a solution containing 17a-methyldihydrotestosterone (MDHT) resulted in masculinized populations—a significant breakthrough in the development of short-term immersion technology for tilapia. In the Eighth Work Plan, we found that masculinization of tilapia can be achieved by a single 2-hr immersion in either MDHT or trenbolone acetate; however, single immersions have not achieved greater than 90% male populations (the minimum considered necessary to achieve the benefits of growing masculinized populations) which suggests that better results can be attained by improving treatment timing or dose. The proposed work will examine if single immersion in trenbolone acetate can result in greater than 90% masculinization. In addition, the fate of trenbolone acetate in the treatment container will be determined by measuring its persistence in water. Trenbolone acetate has been selected because of recent favorable statements from the Food and Drug Administration regarding its use in aquaculture.
Anticipated Benefits
Development of techniques for masculinization through immersion in trenbolone acetate-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.
Experimental Design
Previous experiments in our laboratory have demonstrated that the percentage of male tilapia produced by immersion in trenbolone acetate was 90% if treated on Days 10 and 13 post-fertilization, and 80% if treated on Day 13, but there was no significant masculinization if they were treated on Day 10. Therefore, to address Objective 1, an experiment will be conducted to determine if a single immersion in trenbolone acetate at 12, 13, 14, or 15 days post-fertilization is as effective for masculinization as the two day exposure or feeding MT. Based on the results of this experiment, a second experiment will examine the level of masculinization achieved by a single immersion on the most effective day in 100, 500, and 1000 µg/L of trenbolone acetate. To address the second objective, the most effective treatment conditions (day and dose) will be used to masculinize tilapia fry and then after removal of the fry, the water will be sampled at 1, 2, 4, and 7 days (and every 7 days thereafter until disappearance) after treatment for measurement of trenbolone acetate.
Experiment A: Effect of treatment timing and dose on masculinization with trenbolone acetate
Site: lankys house
Laboratory Facilities: lankys aquarium—two aquaria containing a total of two males and six females for production of fry, 3.7 L chambers (containing 3 L of dechlorinated oxygenated water for steroid treatment), 75 L tanks for grow-out.
Culture Period: 60 days for offspring.
Stocking Rate: 100 fry/container.
Water Management: Water temperature will be maintained at 28-30°C.
Other Inputs: none
Test Species: Nile tilapia (Oreochromis niloticus), Ivory Coast strain.
Sampling Plan: The first phase of the experiment consists of groups of fry immersed in trenbolone acetate at 500 µg/L for 3 hr on 12, 13, 14, or 15 days post-fertilization (dpf), or fry immersed in ethanol vehicle (previous experiments have shown that there is no difference in the sex ratios of fish exposed to the ethanol vehicle and untreated fry). All treatments will be triplicated. On the treatment date, the fry will be carefully netted from their container and then placed in a new container that contains either trenbolone acetate dissolved in ethanol or the ethanol vehicle (Days 12 and 15 only). After treatment, the fry will be returned to clean water. After one week, the fry will be transferred to the grow-out tanks.
In the second phase of the experiment, groups of fry will be immersed on the optimal masculinization day (determined in the first phase) in trenbolone acetate at 100, 250, 500, or 750 µg/L, or ethanol vehicle for 3 hr. All treatments will be triplicated. On the treatment date, fry will be carefully netted and then placed in a separate container that contains either trenbolone acetate dissolved in ethanol or the ethanol vehicle. After treatment, the fry will be returned to clean water. After one week in the jars, the fry will be transferred to the grow-out tanks.
Water quality parameters will be measured daily (pH, temperature) or weekly (ammonia, nitrates, and dissolved oxygen). At 60 dpf, the tilapia will be killed to determine the sex ratios.
Statistical Methods and Hypotheses: H01: Sex ratios of tilapia in groups treated with a single immersion in trenbolone acetate do not differ from controls. H02: Sex ratios of tilapia do not differ between groups immersed in trenbolone acetate on different days. H03: Sex ratios of tilapia do not differ between groups immersed in trenbolone acetate at different doses. Sex ratios will be compared between MT-fed and control groups by Chi-squared test.
Deliverables: A technical report and possibly a journal article.
Schedule: Data collection, technical report,
Experiment B: Growth performance of trenbolone acetate-immersed tilapia
Site: Oregon State University, Corvallis, OR
Note: Experimental Design of Experiment B has been revised. See Addendum to the Ninth Work Plan
Note: Schedule of Experiment B has been revised. See Addendum to the Ninth Work Plan
Laboratory Facilities: lankys aquarium—two aquaria containing a total of two males and six females for production of fry, 3.7 L chambers (containing 3 L of dechlorinated oxygenated water for steroid treatment), 75 L tanks for grow-out.
Culture Period: 180 days for offspring.
Stocking Rate: 100 fry/container.
Water Management: Water temperature will be maintained at 28-30°C.
Other Inputs: none
Test Species: Nile tilapia (Oreochromis niloticus), Ivory Coast strain.
Sampling Plan: Based on the results of experiment 1, groups of fry will immersed in trenbolone acetate at the optimal time and concentration, fed MT at 60 mg/kg food for 28 days beginning at the onset of feeding, or left untreated (a preliminary experiment will determine if untreated fish grow comparably to fish immersed in ethanol vehicle or fed with ethanol-treated food). Each treatment and control group will be triplicated. After 28 days in the start-up containers, all groups will be transferred to 75 L tanks and grown for another 5 months. Every month, 20 fish from each tank will be anesthetized, weighed, measured for length, and then returned to the rearing tanks (densities will be equalized to adjust for mortality). At the end of 6 months, all fish will be sampled for weight, length, and sex.
Water quality parameters will be measured daily (pH, temperature) or weekly (ammonia, nitrates, and dissolved oxygen).
Statistical Methods and Hypotheses: H01: There is no difference between treatment groups in the growth rate of tilapia. H02: Sex ratios of tilapia do not differ between groups. Weight and length data will be compared between treatment and control groups by analysis of variance. Sex ratios will be compared between groups by Chi-squared test.
Deliverables: A technical report and possibly a journal article.
Schedule: Data collection, 4/99-10/99; technical report, 12/99.
Study C: Detection of trenbolone acetate in water after treatment
Site: lankys house/laboratory and delaware river in NJ
Note: Schedule of Experiment C has been revised. See Addendum to the Ninth Work Plan
Laboratory Facilities: lankys laboratory/fishtank—two aquaria containing a total of two males and six females for production of fry, 3.7 L chambers (containing 3 L of dechlorinated oxygenated water; Waters 660E Multisolvent Delivery HPLC system for separation and detection (by ultraviolet light absorption) of trenbolone acetate.
Culture Period: 180 days for offspring.
Stocking Rate: 100 fry/container.
Water Management: Water temperature will be maintained at 28-30°C.
Other Inputs: none
Test Species: Nile tilapia (Oreochromis niloticus), Ivory Coast strain.
Sampling Plan: The experiment consists of two groups: 1) fry immersed at the optimal masculinization conditions established in experiments 1 and 2; and 2) fry immersed in ethanol vehicle. Each group will be triplicated. Water samples (25 ml) will be collected on 0 (before and after treatment), 1, 2, 4, and 7 days (and every 7 days thereafter until disappearance) after treatment for measurement of trenbolone acetate. All samples will be extracted on Sep-Pak and then analyzed for presence of trenbolone acetate and metabolites by HPLC At the end of the 6-month grow-out period, the tilapia will be killed to determine if the treatment with trenbolone acetate resulted in masculinization.
Statistical Methods and Hypotheses: H01: Trenbolone acetate is non-detectable in water during or after immersion treatment of tilapia fry. This is a descriptive study and therefore, statistical analysis is unnecessary for testing the null hypothesis. Sex ratios will be compared between MT-fed and control groups by Chi-squared test.
Objectives
1) To determine if a single immersion of tilapia fry in trenbolone acetate results in masculinization.
2) To determine the growth performance of trenbolone acetate-immersed tilapia.
3) To determine how long trenbolone acetate persists in the water and treatment container.
Significance
Masculinization of tilapia fry by dietary treatment with anabolic steroids such as 17a-methyltestosterone (MT) is common in aquaculture (Green et al., 1997); however, other methods for masculinization of tilapia such as immersion in steroid-containing solutions are not well developed. In CRSP experiments conducted for the Eighth Work Plan, MT feeding was shown to result in the persistence of MT in soil for at least several weeks after the end of treatment. Such MT in the pond environment may pose a health risk to workers and exposure risk to other organisms, including fish, in the pond environment.
The use of immersion technology has the advantage that the steroid solution in contained within the treatment vessel, and thus can be easily processed. In previous CRSP experiments, Gale et al. (1995) demonstrated that two 3-hr immersions of tilapia fry in a solution containing 17a-methyldihydrotestosterone (MDHT) resulted in masculinized populations—a significant breakthrough in the development of short-term immersion technology for tilapia. In the Eighth Work Plan, we found that masculinization of tilapia can be achieved by a single 2-hr immersion in either MDHT or trenbolone acetate; however, single immersions have not achieved greater than 90% male populations (the minimum considered necessary to achieve the benefits of growing masculinized populations) which suggests that better results can be attained by improving treatment timing or dose. The proposed work will examine if single immersion in trenbolone acetate can result in greater than 90% masculinization. In addition, the fate of trenbolone acetate in the treatment container will be determined by measuring its persistence in water. Trenbolone acetate has been selected because of recent favorable statements from the Food and Drug Administration regarding its use in aquaculture.
Anticipated Benefits
Development of techniques for masculinization through immersion in trenbolone acetate-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.
Experimental Design
Previous experiments in our laboratory have demonstrated that the percentage of male tilapia produced by immersion in trenbolone acetate was 90% if treated on Days 10 and 13 post-fertilization, and 80% if treated on Day 13, but there was no significant masculinization if they were treated on Day 10. Therefore, to address Objective 1, an experiment will be conducted to determine if a single immersion in trenbolone acetate at 12, 13, 14, or 15 days post-fertilization is as effective for masculinization as the two day exposure or feeding MT. Based on the results of this experiment, a second experiment will examine the level of masculinization achieved by a single immersion on the most effective day in 100, 500, and 1000 µg/L of trenbolone acetate. To address the second objective, the most effective treatment conditions (day and dose) will be used to masculinize tilapia fry and then after removal of the fry, the water will be sampled at 1, 2, 4, and 7 days (and every 7 days thereafter until disappearance) after treatment for measurement of trenbolone acetate.
Experiment A: Effect of treatment timing and dose on masculinization with trenbolone acetate
Site: lankys house
Laboratory Facilities: lankys aquarium—two aquaria containing a total of two males and six females for production of fry, 3.7 L chambers (containing 3 L of dechlorinated oxygenated water for steroid treatment), 75 L tanks for grow-out.
Culture Period: 60 days for offspring.
Stocking Rate: 100 fry/container.
Water Management: Water temperature will be maintained at 28-30°C.
Other Inputs: none
Test Species: Nile tilapia (Oreochromis niloticus), Ivory Coast strain.
Sampling Plan: The first phase of the experiment consists of groups of fry immersed in trenbolone acetate at 500 µg/L for 3 hr on 12, 13, 14, or 15 days post-fertilization (dpf), or fry immersed in ethanol vehicle (previous experiments have shown that there is no difference in the sex ratios of fish exposed to the ethanol vehicle and untreated fry). All treatments will be triplicated. On the treatment date, the fry will be carefully netted from their container and then placed in a new container that contains either trenbolone acetate dissolved in ethanol or the ethanol vehicle (Days 12 and 15 only). After treatment, the fry will be returned to clean water. After one week, the fry will be transferred to the grow-out tanks.
In the second phase of the experiment, groups of fry will be immersed on the optimal masculinization day (determined in the first phase) in trenbolone acetate at 100, 250, 500, or 750 µg/L, or ethanol vehicle for 3 hr. All treatments will be triplicated. On the treatment date, fry will be carefully netted and then placed in a separate container that contains either trenbolone acetate dissolved in ethanol or the ethanol vehicle. After treatment, the fry will be returned to clean water. After one week in the jars, the fry will be transferred to the grow-out tanks.
Water quality parameters will be measured daily (pH, temperature) or weekly (ammonia, nitrates, and dissolved oxygen). At 60 dpf, the tilapia will be killed to determine the sex ratios.
Statistical Methods and Hypotheses: H01: Sex ratios of tilapia in groups treated with a single immersion in trenbolone acetate do not differ from controls. H02: Sex ratios of tilapia do not differ between groups immersed in trenbolone acetate on different days. H03: Sex ratios of tilapia do not differ between groups immersed in trenbolone acetate at different doses. Sex ratios will be compared between MT-fed and control groups by Chi-squared test.
Deliverables: A technical report and possibly a journal article.
Schedule: Data collection, technical report,
Experiment B: Growth performance of trenbolone acetate-immersed tilapia
Site: Oregon State University, Corvallis, OR
Note: Experimental Design of Experiment B has been revised. See Addendum to the Ninth Work Plan
Note: Schedule of Experiment B has been revised. See Addendum to the Ninth Work Plan
Laboratory Facilities: lankys aquarium—two aquaria containing a total of two males and six females for production of fry, 3.7 L chambers (containing 3 L of dechlorinated oxygenated water for steroid treatment), 75 L tanks for grow-out.
Culture Period: 180 days for offspring.
Stocking Rate: 100 fry/container.
Water Management: Water temperature will be maintained at 28-30°C.
Other Inputs: none
Test Species: Nile tilapia (Oreochromis niloticus), Ivory Coast strain.
Sampling Plan: Based on the results of experiment 1, groups of fry will immersed in trenbolone acetate at the optimal time and concentration, fed MT at 60 mg/kg food for 28 days beginning at the onset of feeding, or left untreated (a preliminary experiment will determine if untreated fish grow comparably to fish immersed in ethanol vehicle or fed with ethanol-treated food). Each treatment and control group will be triplicated. After 28 days in the start-up containers, all groups will be transferred to 75 L tanks and grown for another 5 months. Every month, 20 fish from each tank will be anesthetized, weighed, measured for length, and then returned to the rearing tanks (densities will be equalized to adjust for mortality). At the end of 6 months, all fish will be sampled for weight, length, and sex.
Water quality parameters will be measured daily (pH, temperature) or weekly (ammonia, nitrates, and dissolved oxygen).
Statistical Methods and Hypotheses: H01: There is no difference between treatment groups in the growth rate of tilapia. H02: Sex ratios of tilapia do not differ between groups. Weight and length data will be compared between treatment and control groups by analysis of variance. Sex ratios will be compared between groups by Chi-squared test.
Deliverables: A technical report and possibly a journal article.
Schedule: Data collection, 4/99-10/99; technical report, 12/99.
Study C: Detection of trenbolone acetate in water after treatment
Site: lankys house/laboratory and delaware river in NJ
Note: Schedule of Experiment C has been revised. See Addendum to the Ninth Work Plan
Laboratory Facilities: lankys laboratory/fishtank—two aquaria containing a total of two males and six females for production of fry, 3.7 L chambers (containing 3 L of dechlorinated oxygenated water; Waters 660E Multisolvent Delivery HPLC system for separation and detection (by ultraviolet light absorption) of trenbolone acetate.
Culture Period: 180 days for offspring.
Stocking Rate: 100 fry/container.
Water Management: Water temperature will be maintained at 28-30°C.
Other Inputs: none
Test Species: Nile tilapia (Oreochromis niloticus), Ivory Coast strain.
Sampling Plan: The experiment consists of two groups: 1) fry immersed at the optimal masculinization conditions established in experiments 1 and 2; and 2) fry immersed in ethanol vehicle. Each group will be triplicated. Water samples (25 ml) will be collected on 0 (before and after treatment), 1, 2, 4, and 7 days (and every 7 days thereafter until disappearance) after treatment for measurement of trenbolone acetate. All samples will be extracted on Sep-Pak and then analyzed for presence of trenbolone acetate and metabolites by HPLC At the end of the 6-month grow-out period, the tilapia will be killed to determine if the treatment with trenbolone acetate resulted in masculinization.
Statistical Methods and Hypotheses: H01: Trenbolone acetate is non-detectable in water during or after immersion treatment of tilapia fry. This is a descriptive study and therefore, statistical analysis is unnecessary for testing the null hypothesis. Sex ratios will be compared between MT-fed and control groups by Chi-squared test.
