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Studies on Fate of Methyltestosterone and its Metabolites in Tilapia and on the Use of Phytochemicals as an Alternative Method to Produce a Monosex Population of Tilapia
Reproduction Control Research 1 (10RCR1)/Experiment/Mexico
Universidad Juarez Autonoma de Tabasco, Mexico
Gabriel Márquez Couturier
The Ohio State University
Mary Ann Abiado
1) Determine concentration of methyltestosterone derivatives in tilapia and water using radioimmunoassay and HPLC methods.
2) Evaluate potential action of phytochemicals on sex differentiation of tilapia.
In tilapia aquaculture, all-male populations are desirable because males demonstrate superior growth characteristics compared to females. Moreover, culture of monosex populations prevents reproduction and results in uniformity of fish size. The synthetic steroid, 17a-methyltestosterone (MT) is a derivative of a male specific hormone commonly used to masculinize tilapia juveniles (Green et al., 1997; Abucay and Mair, 1997; Gale et al., 1999). The effect of MT is dependent on various factors such as dose, timing and duration of treatment, and mode of administration (Mirza and Shelton, 1988).
The uptake and depletion of MT have been reported in several species of teleost fish, including cichlids (Fagerlund and Dye, 1979; Goudie et al., 1986; Curtis et al., 1991; Cravedi et al., 1993, Rinchard et al., 1999). As MT administrated orally is readily metabolized, research on the fate of MT and on its metabolites need to be addressed for human and environmental safety issues. In the Tenth Work Plan, we propose to determine the levels of MT and its metabolites in tilapia and water using radioimmunoassay (Rinchard et al., 1999) and high-performance liquid chromatography/mass spectrometry (Williams et al., 2000) methods. The MT antiserum that we used for RIA cross-reacted with testosterone (12.6%), dihydrotestosterone (2.8%) and also with some metabolites of MT identified by Cravedi et al. (1993) such as 17a-methyl-5bandrostan-17b-ol-3-one (48%) and 17a-methyl-5aandrostan-3-17b-diol (12.6%). However, cross-reactivities with circulating androgens are thought to have little impact on our estimates of MT concentrations because the concentration of MT in the plasma of fish not supplemented with MT was negligible (Rinchard et al., 1999).
Another problem associated with the use of MT is that, at high doses or prolonged treatment, MT induces gonadal intersexuality and paradoxical feminization (Goudie et al., 1983; Solar et al., 1984; Van den Hurk et al., 1989; Blasquez et al., 1995; Rinchard et al., 1999; Papoulias et al., 2000). Piferrer and Donaldson (1989) suggested that paradoxical feminization might be due more to aromatization than to inhibition of in vivo synthesis of androgens. However, these authors stressed that in some species aromatization and inhibition of in vivo synthesis of androgens could be the causative factor. Therefore alternative methods to produce monosex populations should be considered.
One approach may involve the use of plant extracts. Gastric intubation of aqueous extracts of Hibiscus macranthus and Basella alba in rat had anabolizing and virilizing effects (Moundipa et al., 1999). Isoflavonoids such as genistein act as estrogen agonists via estrogen receptors in cultured cells and also manifest estrogen-like effects in the female reproductive system (Miksicek, 1995; Santell et al., 1997). However, Levy et al. (1995) reported that following genistein treatment of rats during early pregnancy, the number of males was higher than females among the progenies although the sex ratios were not different that in control. Some flavonoids, such as chrysin, are natural aromatase inhibitors (Chen et al., 1997) and may be used to boost low levels of testosterone in aging males. Results of using tamoxifen, an aromatase inhibitor, in tilapia are controversial. Hines and Watts (1995) are the only authors to report a masculinizing effect of this compound (100 mg/kg of food) in a hybrid tilapia. In the Tenth Work Plan, we intend to evaluate the potential action of different phytochemicals, including genistein, quercetin, gossypol, and oleanolic glycoside on sex differentiation of tilapia. Our laboratory is able to isolate and quantify those phytochemicals in fish tissues using a high-performance liquid chromatography method.
Quantified Anticipated Benefits
The assessment of MT and MT metabolites persistence and fate in tilapia and water will provide information on the potential risks of using MT-feeding technology to produce all-male populations. The use of phytochemicals as an alternative method to produce monosex populations of tilapia will address human and environmental safety issues. Fish offered to the consumer will not be treated with MT and producers may have an alternative method for producing all-male populations of tilapia based on natural products, which do not require FDA approval.
Location of Work: The feeding experiments will be performed both at the Laboratory of Aquaculture, Universidad Juárez Autónoma de Tabasco, Mexico, and at the School of Natural Resources, The Ohio State University, Columbus, Ohio. The experimental diets as well as RIA and HPLC analysis will be carried out at The Ohio State University. The Aquaculture laboratory at the Ohio State University has extensive experience on nutrition, reproduction and sex reversal in tilapia (Mbahinzireki, 2000). We continue to work on phytochemicals in fish diets and their metabolism (Lee et al., 2001; Dabrowski et al., 2001). Over the years, several experiments in our laboratory involved the use of MT and monitoring concentration of this chemical in fish tissues (Rinchard et al., 1999).
Determination of Methyltestosterone Metabolites Concentration in Tilapia Flesh and Water
The experiment will be conducted on first feeding of genetically all-female tilapia Oreochromis niloticus. All-female tilapia populations will be obtained by fertilization of normal eggs with sperm from a phenotypic male genotypically female (XX chromosome set) (Guigen et al., 1999). Fish will be assigned randomly into six tanks (300 fish/tank) and fed ad libitum (3 tanks/dietary treatment) for 30 days. MT will be dissolved in ethyl alcohol and incorporated into a commercial diet at a dose of 40 mg/kg (Abucay and Mair, 1997). Control food will also be treated with ethyl alcohol. Water quality will be monitored throughout the feeding experiment. Temperature and dissolved oxygen will be determined on a daily basis with biweekly measurements of total ammonia-nitrogen and pH. Every 10 days, fish and water will be sampled (n=15) from each tank and frozen for further analysis. At the end of the experiment, growth performance and survival will be evaluated. Sex ratio will be determined by microscopic analysis of gonadal squashes (Guerrero and Shelton, 1974; Guigen et al., 1999). At that stage ovaries are characterized by the presence of oocytes easily identifiable in their auxocytosis or previtellogenic stages, whereas testes are characterized by their typical lobular configuration (Nakamura and Nagahama, 1985). MT and MT metabolites in fish tissues and water will be determined using RIA (Rinchard et al., 1999) and HPLC (Williams et al., 2000) following extraction.
Evaluation of Potential Action of Phytochemicals on Sex Differentiation of Tilapia
The experiment will be conducted on first feeding genetically all-female tilapia Oreochromis niloticus. Fish will be randomly distributed into 21 aquaria at a density of 100 fish per aquarium with three replicates per treatment. One hundred larvae will be weighed and frozen for subsequent analysis of whole body composition. Fish will be fed at a restricted ration up to 90% satiation for 4-6 weeks. Semi-purified diets will be formulated based on our previous experience (Lee and Dabrowski, 2001). Semi-purified diets will be used because they are least contaminated with natural steroids (Feist and Schreck, 1990). In order to determine the effects of phytochemicals on sex differentiation, seven casein-gelatin based diets will be prepared. Diet 1 (negative control) will be free of phytochemicals. Diets 2 and 3 (positive controls) will contain either MT at a dose of 40 mg/kg (Abucay and Mair, 1997) or ATD (1,4,6-androstratiene-3-17-dione; aromatase inhibitor) at an initial dose of 150 mg/kg (Guigen et al., 1999). Diets 4 to 7 will contain gossypol, genistein, quercetin and oleanolic glycoside at a dose of 500 mg/kg. The amounts correspond to the levels commonly found in seed meals of plants (Benneteau-Pelissero et al., 2001; Dabrowski and Lee, 2001). In the second year of study concentration of phytochemicals will be modified. Water quality will be monitored throughout the feeding experiment. Temperature and dissolved oxygen will be determined on a daily basis with biweekly measurements of total ammonia-nitrogen and pH. At the end of the feeding trial, growth performance will be evaluated in terms of final individual body weight, survival (%), specific growth rate (SGR, %) and weight gain (%). Fish from each dietary treatment also will be sampled for proximate analysis (water, protein, lipid, ash) and phytochemicals analysis (Dabrowski et al., 2001). Sex ratio will be determined as previously described (F.2.1).
Statistical Analysis: Analyses will be performed using the Statistical Analysis System (SAS Institute, Inc., Cary, NC). Data on growth performance and survival will be subjected to one-way analysis of variance (ANOVA) followed by a comparison of means using Scheffe's F test (Dagnelie, 1975). The Chi-square test will be used to determine alterations in sex ratios. Normality and homogeneity of variance tests will be performed on raw data. Sample distributions violating assumptions will be log-transformed before analysis. Data, expressed as percentages, will be arc sine-transformed before analysis. All differences will be regarded as significant at P < 0.05.
Tabasco is considered among the States of Mexico with the highest potential for both intensive and extensive aquacultural development. Moreover, fish consumption constitutes an important part of the rural lifestyle in the State of Tabasco. Therefore, research efforts being proposed are logical initial steps toward developing sustainable aquaculture in the region. The research will benefit the entire region by providing pertinent information on masculinization of tilapia using natural phytochemicals.
Determination of Methyltestosterone Metabolites Concentration in Tilapia and Water
July-August 2001, preparation of the experimental diets (MT and control);
September 2001, production of all-female tilapia population;
September-October 2001, feeding experiment and sampling;
November 2001-July 2002, measurement of MT and metabolites in tilapia and water using radioimmunoassay and high-performance liquid chromatography/mass spectrometry;
July-December 2002, data analysis and preparation of reports and publications.
Evaluation of Potential Action of Phytochemicals on Sex Differentiation of Tilapia
October 2001, formulation and preparation of the experimental diets
November 2001, production of all-female tilapia population;
December 2001-February 2002, first set of feeding experiments and sampling;
March-April 2002, measurement of phytochemicals in tilapia tissue;
May 2002, production of all-female tilapia population;
June-July 2002, second set of feeding experiments and sampling
August-September, measurement of phytochemicals in tilapia tissue;
October-December 2002, data analysis and preparation of reports and publications.
Final Report: 30 April 2003
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