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Juicing my Gold fish
- Thread starter bigjd69
- Start date
I
immortalis
Guest
haha, this shit is gettin outta control p~
lanky
Well-known member
Objective
To determine the optimal treatment conditions for masculinization of tilapia(it is a very tasty fish) 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
Laboratory Facilities: lankys aquarium 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 28-30oC.
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.
Effect of Fish Density on Efficacy of Masculinization by Immersion in MDHT
(Reproduction Control Research 2B)
Laboratory Facilities: 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 28-30oC.
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.
Masculinization of Tilapia Fry by Immersion in MDHT at a Production Level
(Reproduction Control Research 2C)
Pond Facilities: Auburn University 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.
YES YOUR FISH CAN DRINK WINNY! lol
To determine the optimal treatment conditions for masculinization of tilapia(it is a very tasty fish) 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
Laboratory Facilities: lankys aquarium 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 28-30oC.
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.
Effect of Fish Density on Efficacy of Masculinization by Immersion in MDHT
(Reproduction Control Research 2B)
Laboratory Facilities: 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 28-30oC.
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.
Masculinization of Tilapia Fry by Immersion in MDHT at a Production Level
(Reproduction Control Research 2C)
Pond Facilities: Auburn University 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.
YES YOUR FISH CAN DRINK WINNY! lol
lanky
Well-known member
ohh..i did not type that one..it is a copy and paste job from a real study....i have some on trenbolone and methyltestosterone on fish masculinization..
tried to get one on silverback gorilla's on trenbolone and halotestin...unfortunatly the researchers did not survive past the third medication administration of Halo..jk
tried to get one on silverback gorilla's on trenbolone and halotestin...unfortunatly the researchers did not survive past the third medication administration of Halo..jk
lanky
Well-known member
YOUR FISH ON TRENBOLONE...
Previous experiments in our laboratory have demonstrated that short-term immersions in synthetic steroids such as 17-methyldihydrotestosterone (MDHT) and trenbolone acetate (TA) are effective masculinizing treatments for Nile tilapia fry (Contreras et al., 1997, 1999, 2000; Gale et al., 1999). Recently, Bart et al. (2000) have found that ultrasound treatment enhances masculinization when short-term immersions are used. However, short-term immersions are far from being used on a commercial scale. One of the major criticisms to this method is that the concentration used is higher than the amount of 17-methyltestosterone (MT) used in feeding trials. However, the immersion protocol presents advantages over feeding treatment for hormone control and disposal, as well as fewer risks of environmental contamination.
The use of TA in the cattle industry for growth enhancement has created expectations in the aquaculture industry. This steroid can be used for androgenic purposes and may have anabolic effects while fry are under masculinization treatment. It has been reported that TA administered in the food successfully masculinized channel catfish (Galvez et al., 1995) andblue tilapia (Galvez et al., 1996). However, Davis et al. (2000) found that the catfish treated with TA were not functional males but infertile organisms after three years of growth.
Research is needed to understand how much steroid is available to the fry during immersion treatment. Because of their hydrophobic properties, steroids may be very unstable in the water treatment, form precipitates, or bind to the walls of the containers. To address some of these questions, we performed experiments to determine the concentration of TA in dechlorinated water and distilled water at different times.
Methods and Materials
Experiment 1: Steroid Solutions without Fry Immersed
Following an experiment reported on in Contreras et al. (2001) involving examination of TA solution following immersion of fry, a similar experiment with no fry immersion was planned. This experiment consisted of three 3.8-l glass jars containing 1 l of well water. To each replicate 500 ml of TA were added, mixed thoroughly, and maintained at 28 ± 1°C under constant aeration. Samples were collected at 0, 3, and 30 h at the surface, middle, and bottom of the jar (samples taken at 30 hours at middle depth were lost during processing).
Experiment 2: Short Trial in Borosilicate Tube
Ten ml of double-distilled water were placed in a borosilicate tube, to which 100 ml of TA were added and thoroughly mixed by vortexing. Samples of 1 ml were collected at time 0 (from the middle of the tube) and at 1 h (from the surface, middle, and bottom). Once all samples were collected, the water remaining in the tube was vortexed, and the tube was emptied and washed with 1 ml of ether to assay for TA attached to the tube walls.
TA Detection
All samples were stored frozen (–20°C) until processed for TA detection. From each sample, 2.0 ml were extracted in 8 ml of diethyl ether. The organic phase of each sample was collected in a new tube after the aqueous phase was snap-frozen in liquid nitrogen. The extraction procedure was repeated, and the ether extracts were pooled for each sample and dried down in a SpeedVac. Each dried extract was reconstituted in 1 ml of methanol. Aliquots of the reconstituted extracts were removed to 150-ml glass inserts for determination of TA concentration by High Performance Liquid Chromatography (HPLC). The HPLC methods followed the procedure outlined in Huang et al. (1983) and modified by Feist et al. (1990). The HPLC analysis was performed using a Waters System consisting of a 600 controller, 717 autosampler, 996 photodiode array detector, a Dell Dimension V400c computer, Millenium PDA software, and a reverse phase C18 column (flow rate 0.4 ml min-1). We used an isocratic mobile phase of water:methanol:acetonitrile: isopropanol (62:28:5:5) followed by a linear gradient (3.3% min-1) of water:methanol:butanol (35:45:20) for 30 min monitored at a wide variety of wavelengths but specifically analyzed at 254, 280, and 340 nm. This system allows for the separation of 19 steroid standards with detection limits of 3 ng for each steroid. Each sample was analyzed once.
Results
Experiment 1
Concentrations of TA in well water were variable at all times and at all sampling points (surface, middle, and bottom). At time 0, levels of TA in the surface water were lower than the expected value (mean = 300.7 mg l-1, SD = 87.5). Initial values of hormone concentration range from 211.2 to 372.4 mg l-1. Similar patterns were observed at 3 and 30 h after addition of the steroid. This trend was also observed in samples taken at the middle point and bottom of the jars (means = 279.5 and 271.8mg l-1, respectively). Concentration values of TA showed no consistent patterns (Figure 1).
Experiment 2
Concentration of TA at time 0 was 8,731.5 mg ml-1 (the expected value was 10,000 mg ml-1). No significant changes were observed in the samples taken at the surface, middle, or bottom of the tube after one hour of mixing of the steroid with water (Figure 2). TA was detected in the ether used for rinsing the glass tube (1,887.7 mg l-1). The estimated total amount of TA detected in the borosilicate tube after adding the concentration rinsed from the glass accounted for 92.4% of the total amount of TA added to the tube.
Discussion
Our earlier findings (Contreras et al., 2001) indicate that the target dose for TA immersion is rarely achieved. The surprisingly low levels of TA found in our previous report have forced us to validate these results by conducting further experiments. However, we have found that the patterns are maintained independently of the source of water used (dechlorinated versus well water) or the location at which the samples are collected (surface, middle, or bottom of jars). Our first hypothesis focused on the precipitation of TA out of solution after mixing with water. However, the data from the experiments reported here indicate that samples from the bottom of the jar have similar patterns to those observed in the surface water. Another hypothesis for explaining the low levels of steroid in the water is that the steroid could be binding to the walls of the jars (which may be porous). The data obtained from a borosilicate tube (used for assays because of its low-binding properties) indicate that about 2% of the TA added binds to the glass. Therefore, it can be expected that binding to jars is higher. More research is needed to determine if the glass employed is trapping a significant amount of steroid, decreasing the efficacy of the immersion technique.
Anticipated Benefits
Masculinizing Nile tilapia fry by immersion can be a good alternative to feeding the fry with hormone-impregnated food. However, the amount of variability observed in the concentration of the steroid used during treatment indicates that this technique requires refinement to obtain more consistent results.
Literature Cited
Bart, A., A.R.S.B. Athauda, and J.S. Diana, 2001. The application of ultrasound to produce all-male tilapia using immersion protocol. In: A. Gupta, K. McElwee, D. Burke, J. Burright, X. Cummings, and H. Egna (Editors), Eighteenth Annual Technical Report. Pond Dynamics/Aquaculture CRSP, Oregon State University, Corvallis, Oregon, p. 55.
Contreras Sánchez, W.M., M.S. Fitzpatrick, and C.B. Schreck, 2001. Masculinization of tilapia by immersion in trenbolone acetate: Detection of trenbolone acetate after treatment. In: A. Gupta, K.McElwee, D. Burke, J. Burright, X. Cummings, and H. Egna (Editors), Eighteenth Annual Technical Report. Pond Dynamics/Aquaculture CRSP, Oregon State University, Corvallis, Oregon, pp. 47–49.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, R.H. Milston, and C.B.Schreck, 1997. Masculinization of Nile tilapia (Oreochromis niloticus) by single immersion in 17-methyltestosterone and trenbolone acetate. In: K. Fitzsimmons (Editor), Proceedings of the Fourth International Symposium on Tilapia Aquaculture. Orlando, Florida, pp. 783–790.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, R.H. Milston, and C.B.Schreck, 1999. Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in synthetic androgens: Timing and efficacy. In: B.W. Green, H.C. Clifford, M. McNamara, G. Montaño-Moctezuma (Editors), Proceedings of the Fifth Central American Symposium on Aquaculture. San Pedro Sula, Honduras, pp. 246–248.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, R.H. Milston, and C.B.Schreck, 2000. Masculinization of Nile tilapia with steroids: Alternative 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 Reproductive Physiology of Fish. John Grieg, A.S., Bergen, Norway, pp. 250–252.
Davis, K.B., C.A. Goudie, B.A. Simco, T.R. Tiersch, and G.J. Carmichael, 1992. Influence of dihydrotestosterone on sex determination in channel catfish and blue catfish: Period of developmental sensitivity. Gen. Comp. Endocrinol., 86:147–151.
Feist, G., C.B. Schreck, M.S. Fitzpatrick, and J.M. Redding, 1990. Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation. Gen. Comp. Endocrinol., 80:299–313.
Gale, W.L., M.S. Fitzpatrick, M. Lucero, W.M. Contreras-Sánchez, and C.B. Schreck, 1999. Production of all-male populations of Nile tilapia (Oreochromis niloticus) by immersion in androgens. Aquaculture, 178:349–357.
Galvez, J.I., J.R. Morrison, and R.P. Phelps, 1996. Efficacy of trenbolone acetate in sex inversion of the blue tilapia Oreochromis aureus. J.World Aquacult. Soc., 27:483–486.
Galvez, J.I., P.M. Mazik, R.P. Phelps, and D.R. Mulvaney, 1995. Masculinization of channel catfish Ictalurus punctatus by oral administration of trenbolone acetate. J. World Aquacult. Soc., 26(4):378–383.
Huang F.L., F.C. Ke, J.J. Hwang, and T.B. Lo, 1983. High-pressure liquid chromatography separation of a mixture of corticosteroids, androgens, and progestins. Arch. Biochem. Biophys., 225:512–517
Previous experiments in our laboratory have demonstrated that short-term immersions in synthetic steroids such as 17-methyldihydrotestosterone (MDHT) and trenbolone acetate (TA) are effective masculinizing treatments for Nile tilapia fry (Contreras et al., 1997, 1999, 2000; Gale et al., 1999). Recently, Bart et al. (2000) have found that ultrasound treatment enhances masculinization when short-term immersions are used. However, short-term immersions are far from being used on a commercial scale. One of the major criticisms to this method is that the concentration used is higher than the amount of 17-methyltestosterone (MT) used in feeding trials. However, the immersion protocol presents advantages over feeding treatment for hormone control and disposal, as well as fewer risks of environmental contamination.
The use of TA in the cattle industry for growth enhancement has created expectations in the aquaculture industry. This steroid can be used for androgenic purposes and may have anabolic effects while fry are under masculinization treatment. It has been reported that TA administered in the food successfully masculinized channel catfish (Galvez et al., 1995) andblue tilapia (Galvez et al., 1996). However, Davis et al. (2000) found that the catfish treated with TA were not functional males but infertile organisms after three years of growth.
Research is needed to understand how much steroid is available to the fry during immersion treatment. Because of their hydrophobic properties, steroids may be very unstable in the water treatment, form precipitates, or bind to the walls of the containers. To address some of these questions, we performed experiments to determine the concentration of TA in dechlorinated water and distilled water at different times.
Methods and Materials
Experiment 1: Steroid Solutions without Fry Immersed
Following an experiment reported on in Contreras et al. (2001) involving examination of TA solution following immersion of fry, a similar experiment with no fry immersion was planned. This experiment consisted of three 3.8-l glass jars containing 1 l of well water. To each replicate 500 ml of TA were added, mixed thoroughly, and maintained at 28 ± 1°C under constant aeration. Samples were collected at 0, 3, and 30 h at the surface, middle, and bottom of the jar (samples taken at 30 hours at middle depth were lost during processing).
Experiment 2: Short Trial in Borosilicate Tube
Ten ml of double-distilled water were placed in a borosilicate tube, to which 100 ml of TA were added and thoroughly mixed by vortexing. Samples of 1 ml were collected at time 0 (from the middle of the tube) and at 1 h (from the surface, middle, and bottom). Once all samples were collected, the water remaining in the tube was vortexed, and the tube was emptied and washed with 1 ml of ether to assay for TA attached to the tube walls.
TA Detection
All samples were stored frozen (–20°C) until processed for TA detection. From each sample, 2.0 ml were extracted in 8 ml of diethyl ether. The organic phase of each sample was collected in a new tube after the aqueous phase was snap-frozen in liquid nitrogen. The extraction procedure was repeated, and the ether extracts were pooled for each sample and dried down in a SpeedVac. Each dried extract was reconstituted in 1 ml of methanol. Aliquots of the reconstituted extracts were removed to 150-ml glass inserts for determination of TA concentration by High Performance Liquid Chromatography (HPLC). The HPLC methods followed the procedure outlined in Huang et al. (1983) and modified by Feist et al. (1990). The HPLC analysis was performed using a Waters System consisting of a 600 controller, 717 autosampler, 996 photodiode array detector, a Dell Dimension V400c computer, Millenium PDA software, and a reverse phase C18 column (flow rate 0.4 ml min-1). We used an isocratic mobile phase of water:methanol:acetonitrile: isopropanol (62:28:5:5) followed by a linear gradient (3.3% min-1) of water:methanol:butanol (35:45:20) for 30 min monitored at a wide variety of wavelengths but specifically analyzed at 254, 280, and 340 nm. This system allows for the separation of 19 steroid standards with detection limits of 3 ng for each steroid. Each sample was analyzed once.
Results
Experiment 1
Concentrations of TA in well water were variable at all times and at all sampling points (surface, middle, and bottom). At time 0, levels of TA in the surface water were lower than the expected value (mean = 300.7 mg l-1, SD = 87.5). Initial values of hormone concentration range from 211.2 to 372.4 mg l-1. Similar patterns were observed at 3 and 30 h after addition of the steroid. This trend was also observed in samples taken at the middle point and bottom of the jars (means = 279.5 and 271.8mg l-1, respectively). Concentration values of TA showed no consistent patterns (Figure 1).
Experiment 2
Concentration of TA at time 0 was 8,731.5 mg ml-1 (the expected value was 10,000 mg ml-1). No significant changes were observed in the samples taken at the surface, middle, or bottom of the tube after one hour of mixing of the steroid with water (Figure 2). TA was detected in the ether used for rinsing the glass tube (1,887.7 mg l-1). The estimated total amount of TA detected in the borosilicate tube after adding the concentration rinsed from the glass accounted for 92.4% of the total amount of TA added to the tube.
Discussion
Our earlier findings (Contreras et al., 2001) indicate that the target dose for TA immersion is rarely achieved. The surprisingly low levels of TA found in our previous report have forced us to validate these results by conducting further experiments. However, we have found that the patterns are maintained independently of the source of water used (dechlorinated versus well water) or the location at which the samples are collected (surface, middle, or bottom of jars). Our first hypothesis focused on the precipitation of TA out of solution after mixing with water. However, the data from the experiments reported here indicate that samples from the bottom of the jar have similar patterns to those observed in the surface water. Another hypothesis for explaining the low levels of steroid in the water is that the steroid could be binding to the walls of the jars (which may be porous). The data obtained from a borosilicate tube (used for assays because of its low-binding properties) indicate that about 2% of the TA added binds to the glass. Therefore, it can be expected that binding to jars is higher. More research is needed to determine if the glass employed is trapping a significant amount of steroid, decreasing the efficacy of the immersion technique.
Anticipated Benefits
Masculinizing Nile tilapia fry by immersion can be a good alternative to feeding the fry with hormone-impregnated food. However, the amount of variability observed in the concentration of the steroid used during treatment indicates that this technique requires refinement to obtain more consistent results.
Literature Cited
Bart, A., A.R.S.B. Athauda, and J.S. Diana, 2001. The application of ultrasound to produce all-male tilapia using immersion protocol. In: A. Gupta, K. McElwee, D. Burke, J. Burright, X. Cummings, and H. Egna (Editors), Eighteenth Annual Technical Report. Pond Dynamics/Aquaculture CRSP, Oregon State University, Corvallis, Oregon, p. 55.
Contreras Sánchez, W.M., M.S. Fitzpatrick, and C.B. Schreck, 2001. Masculinization of tilapia by immersion in trenbolone acetate: Detection of trenbolone acetate after treatment. In: A. Gupta, K.McElwee, D. Burke, J. Burright, X. Cummings, and H. Egna (Editors), Eighteenth Annual Technical Report. Pond Dynamics/Aquaculture CRSP, Oregon State University, Corvallis, Oregon, pp. 47–49.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, R.H. Milston, and C.B.Schreck, 1997. Masculinization of Nile tilapia (Oreochromis niloticus) by single immersion in 17-methyltestosterone and trenbolone acetate. In: K. Fitzsimmons (Editor), Proceedings of the Fourth International Symposium on Tilapia Aquaculture. Orlando, Florida, pp. 783–790.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, R.H. Milston, and C.B.Schreck, 1999. Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in synthetic androgens: Timing and efficacy. In: B.W. Green, H.C. Clifford, M. McNamara, G. Montaño-Moctezuma (Editors), Proceedings of the Fifth Central American Symposium on Aquaculture. San Pedro Sula, Honduras, pp. 246–248.
Contreras-Sánchez, W.M., M.S. Fitzpatrick, R.H. Milston, and C.B.Schreck, 2000. Masculinization of Nile tilapia with steroids: Alternative 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 Reproductive Physiology of Fish. John Grieg, A.S., Bergen, Norway, pp. 250–252.
Davis, K.B., C.A. Goudie, B.A. Simco, T.R. Tiersch, and G.J. Carmichael, 1992. Influence of dihydrotestosterone on sex determination in channel catfish and blue catfish: Period of developmental sensitivity. Gen. Comp. Endocrinol., 86:147–151.
Feist, G., C.B. Schreck, M.S. Fitzpatrick, and J.M. Redding, 1990. Sex steroid profiles of coho salmon (Oncorhynchus kisutch) during early development and sexual differentiation. Gen. Comp. Endocrinol., 80:299–313.
Gale, W.L., M.S. Fitzpatrick, M. Lucero, W.M. Contreras-Sánchez, and C.B. Schreck, 1999. Production of all-male populations of Nile tilapia (Oreochromis niloticus) by immersion in androgens. Aquaculture, 178:349–357.
Galvez, J.I., J.R. Morrison, and R.P. Phelps, 1996. Efficacy of trenbolone acetate in sex inversion of the blue tilapia Oreochromis aureus. J.World Aquacult. Soc., 27:483–486.
Galvez, J.I., P.M. Mazik, R.P. Phelps, and D.R. Mulvaney, 1995. Masculinization of channel catfish Ictalurus punctatus by oral administration of trenbolone acetate. J. World Aquacult. Soc., 26(4):378–383.
Huang F.L., F.C. Ke, J.J. Hwang, and T.B. Lo, 1983. High-pressure liquid chromatography separation of a mixture of corticosteroids, androgens, and progestins. Arch. Biochem. Biophys., 225:512–517
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