AQUACULTURE CRSP 22 NNUAL TECHNICAL REPORTpdacrsp.oregonstate.edu/pubs/technical/22tch/40-11FNFR3.pdf · 2005. 9. 20. · AQUACULTURE CRSP 22ND ANNUAL TECHNICAL REPORT 287 USE OF

AQUACULTURE CRSP 22ND ANNUAL TECHNICAL REPORT

287

USE OF PHYTOCHEMICALS AS AN ENVIRONMENTALLY FRIENDLY METHOD TO SEX REVERSE NILE TILAPIA

Eleventh Work Plan, Fish Nutrition and Feed Technology Research 3 (11FNFR3)Final Report

Konrad Dabrowski, Gustavo Rodriguez, Mary Ann G. AbiadoSchool of Natural Resources

The Ohio State University, Columbus, Ohio

Wilfrido Contreras Sanchez and Maria de Jesus Contreras GarciaUniversidad Juarez Autonoma de Tabasco

Tabasco, Villahermosa, Mexico

Printed as Submitted

ABSTRACT

This study evaluated the use of daidzein, chrysin, and caffeic acid as potential sex reversal agents in Nile tilapia by dietary administration or immersion treatments. Three feeding experiments were conducted at two locations: Aqua-culture Laboratory at the Ohio State University (Experiment 1 and 2) and the Aquaculture laboratory at the Universi-dad Juarez Autonoma de Tabasco UJAT-Mexico (Experiment 3). An immersion treatment experiment took place at the Aquaculture Laboratory of The Ohio State University (Experiment 4). The dietary administration experiments were as follows: for Experiments 1 and 3, diets control, chrysin (500 mg/kg), daidzein (500 mg/kg) and caffeic acid (500 mg/kg) along with the steroidal compounds spironolactone (500 mg/kg), 1,4,6-androstratiene-3-17-dione ATD (150 mg/kg) and 17α-methyltestosterone (MT) (60 mg/kg) were fed for 8 weeks. For Experiment 3, the diets were control, chrysin (500 mg/kg), daidzein (500 mg/kg) and caffeic acid (500 mg/kg) along with the steroidal compounds spi-ronolactone (500 mg/kg) were offered for 6 weeks. In all cases, semi-purified casein gelatin diets were used to avoid contamination with external sources of either phytochemicals or steroid-like compounds. For the immersion experi-ment (Experiment 4), four immersion trials were carried out at 10, 17, 21, and 28 day post-hatching on the following chemicals and concentrations, vehicle DMSO (1 ml/l), daidzein 400 (mg/l), chrysin (20 mg/l), caffeic acid (40 mg/l), spironolactone (5 mg/l), ATD (1.2 mg/l) and MT (400 µg/l). In all experiments, final sex ratio was determined by gonad squash; in feeding trials, the final individual body weight and survival were evaluated. Results of experiments conducted at The Ohio State University indicate that the presence of the tested phytochemicals in food or in immer-sion baths has no effect on the final sex ratio of tilapia or growth performance. In Experiment 1, ATD and MT had a significant effect on final sex ratio (50% and 100% masculinization, respectively); in Experiment 3 (UJAT-Mexico) MT and ATD along with SPIRO affected the male ratio significantly. No effects were observed in Experiment 2 (97±3% males) or Experiment 4 (60-40% male:female ratio) across experimental groups.

INTRODUCTION

The increased use of synthetic steroid hormones to produce monosex populations of tilapia for intensive productive systems may lead to environmental and public health concerns. Such a situation is derived from the fact that female organism of tilapine species have a high fecundity, generally reproducing at a small size and exhibiting stunted somatic growth at higher densi-ties, while male tilapias exhibit faster growth rates and are often the preferred gender for monosex aquaculture (Hines and Watts 1995). The efficacy of MT is dependent on many factors such as dose, timing, and duration of treatment (Mirza and Shelton, 1988), providing another disadvantageous characteristic on the use of hormones

in tilapia aquaculture.

The direct study of the mechanisms of hormone bio-synthesis and action on the gonad sex differentiation process could also provide alternative methods for pro-ducing monosex populations of fish. Among this mecha-nisms, the enzyme aromatase is of particular interest in sexual differentiation in fish and many other organisms. The enzyme is a cytochrome, P-450 hemoprotein, which catalyzes the conversion of androgens to estrogens (Bro-die et al., 1999, Le Bail et al., 1998, Strauss et al., 1998, Griffiths et al., 1999, Eng et al., 2001), a critical stage that affects the sex differentiation process in vertebrates. There is evidence of aromatase inhibition by physical factors, such as temperature (D’cotta et al., 2001, Kitano

TWENTY-SECOND ANNUAL TECHNICAL REPORT288 FISH NUTRITION AND FEED TECHNOLOGY RESEARCH 289

et al., 2000) as well as steroidal and non steroidal chemi-cal compounds (Brodie et al., 1999, Smith, 1999, Seralini and Moslemi, 2001) that have achieved a certain degree of success in sex inversion in fish. Consequently it can be considered that inhibition of aromatase action by physi-cal and chemical factors could mimic the sex-reversal ef-fects of androgen treatments in some fish species (Kwon et al., 2001).

Chemical compounds classified as phytochemicals (isoflavonoids, flavonoids and ligands) are natural steroid-like compounds derived from soy, tea, fruits and vegetables with a reported aromatase inhibition ability that could be able to suppress estrogen biosynthesis in cells (Geahlen et al., 1989, Pelissero et al., 1996; Eng et al., 2001). Such action is facilitated given the stable structure and low molecular weights of phytochemicals that can pass through cell membranes (Ososki and Kenelly, 2003). In general, the action of phytochemicals when adminis-tered to fish is a recent topic of study, the possible effect of flavonoids on fish aromatase caught attention in a nutritional study with female sturgeons, the hormonal profile in those fish fed with a high dietary inclusion of soybean meal; led to a decrease of plasma estrogen levels (Pelissero et al 1996).

Many studies indicate that aromatase inhibition capac-ity of phytochemicals is closely related to their chemical structure (Le Bail et al., 1998, Jeong et al., 1999, Saa-rinen et al., 2001). The degree of hydroxylation on the molecule increases their enzymatic inhibitory ability, therefore a higher number of hydroxyl radicals increases inhibitory activity (Krazeisen et al., 2002). The flavone 7-hydroxyflavone and apineginin used in microsomes of the human placenta after normal full-term delivery were the most effective aromatase and 17β-hydroxysteroid dehydrogenase inhibitors; this experiment showed that flavonoids with 7-methoxy or 8-hydroxyl groups in the ring A showed an important anti-aromatase activity, thus there is an implied structure-activity relation (Le Bail et al., 1998). However is very important to consider that aromatase affinity for flavonoids in generally lower, than it is for steroidal derivatives (Seralini and Moslemi 2001).

A second approach on the use of phytochemicals as endocrine disruptors is their interaction with estrogen nuclear receptors. 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). Flavonoids have been found to act as phytoestrogens since these compounds have structures that are recog-nized as estrogen mimics for the estrogen receptor. They can compete with endogenous estrogens for binding to the estrogen receptor; therefore, they can act as antiestro-gens or weak estrogens (Miyahara et al., 2003). The abil-ity of non-steroidal compounds such as phytoestrogens to bind to the estrogen receptor is partially determined by the distance between the two extreme hydroxyl

groups. Phytoestrogens are usually weak estrogens due to their lower affinity for the estrogen receptors. Whether phytoestrogens act as estrogens depends on the presence and relative concentration of stronger steroidal estrogens (Rickard and Thompson, 1995).

There is a strong variation of the results obtained after using pure phytochemicals and their action in vitro in the inhibition of estrogen synthesis (Joshi et al., 1998). Most of the information available in fish is related to this activity in vitro using gonad cells and measuring the coefficients of inhibitions of synthesis of estrogens when flavonoids are presents at several concentrations. There is not an established knowledge of how flavonoids with steroidal activity are absorbed and metabolized by ani-mals, thus their in vivo effect remains mostly unknown. The present research work is focused on the use of three different phytochemicals to evaluate their potential ac-tion in vivo on sex inversion in tilapia larvae as non ste-roidal endocrine disruptors, simultaneously with other steroidal endocrine disruptor chemicals.

METHODS AND MATERIALS

Study: Evaluation of potential action of potential phy-tochemicals on sex differentiation of tilapia by dietary administration.

A series of experiments were conducted at the Ohio State University (OSU) aquaculture laboratory and UJAT-Mexico. In these experiments the purified phytochemi-cals daidzein, chrysin, and caffeic acid (ICN® Aurora, OH) were supplemented to semi-purified casein gelatin-based diets at a continuous concentration of 500 mg/kg of diet (see Table 1). Also the steroidal compounds 17α-methyltestosterone (MT) (Sigma® St Louis, MO), 1,4,6-androstatrien-3-17-dione or androstatrienedione (ATD) (Steraloids Inc® Newport, RI) and spironolactone (ICN®) for comparison among non-steroidal and steroidal sex reversal agents. For each experiment different genotypes were used to evaluate the potential effect of the men-tioned phytochemicals, each experiment by genotype and location is described as follows:

Experiment 1: All-female tilapia experiment OSU

The experiment was conducted on first feeding tilapia Oreochromis niloticus genetically all-female (Phil-FishGen, Nueva Ecija Philippines) Fish were randomly distrib-uted into glass aquaria (35 l) in a recirculation system at a constant temperature of 26±2 °C, and a density of 60 fish per tank with three replicates per treatment. Experi-mental casein-gelatin based diets were formulated (Table 1) and designated as follows: control (CON), 17α-me-thyltestosterone (MT) 60 mg/kg, androstatrienedione (ATD)150 mg/kg, daidzein (DAID) 500 mg/kg, chrysin (CHR) 500 mg/kg, caffeic acid (CAFF) 500 mg/kg and spironolactone (SPIRO) 500 mg/kg, for the MT and ATD


diets, in both cases the chemicals were dissolved in ethyl ethanol and incorporated to the control diet. Fish were fed at 10-15% body weight ratio for 8 weeks. Growth performance was evaluated in terms of the final indi-vidual mean body weight, survival (%), specific growth rate (SGR, %) and weight gain (%). The final sex was determined by microscopic analysis of gonad squashes at the end of experiment (Guerrero and Shelton, 1974). Partial samples per tank (5 fish) took place at 2, 4, 6, and 8 weeks to establish phytochemicals absorption.

Experiment 2: All-male tilapia experiment OSU

The experiment was conducted on first feeding geneti-cally all-male tilapia (Til-Tech, Robert, LA). Fish were randomly distributed into glass aquaria (35 l) in a recirculation system at a constant temperature of 26±2 °C, and a density of 100 fish per tank with three repli-cates per treatment. Experimental casein-gelatin based diets were used as follows: control (CON), daidzein (DAID) 500 mg/kg, chrysin (CHR) 500 mg/kg, caffeic acid (CAFF) 500 mg/kg and spironolactone (SPIRO) 500 mg/kg. Fish were fed at 10-15% body weight ratio for 6 weeks. Growth performance was evaluated in terms of the final individual body weight, survival (%), specific growth rate (SGR, %) and weight gain (%). The final sex was determined by microscopic analysis of gonad squashes at the end of experiment (Guerrero and Shel-ton, 1974). Fish samples (10 fish) per tank took place at weeks 4 and 6 to establish phytochemicals absorption.

Experiment 3: Mixed sex tilapia UJAT-Mexico

The experiment was conducted on a mixed sex group of first feeding tilapia. Fish were randomly distributed into plastic containers (20 l) with daily water exchanges at a temperature 21.5±2 °C, and density of 150 fish per aquarium with three replicates per treatment. The same experimental casein-gelatin based diets that were used for the all-female OSU experiment were used in this ex-periment as follows: control (CON), 17α-methyltestoster-one (MT), androstatrienedione (ATD), daidzein (DAID), chrysin (CHR), caffeic acid (CAFF) and spironolactone (SPIRO). Fish were fed at 10-15% body weight ratio for 6 weeks. Growth performance was evaluated in terms of the final individual mean body weight and survival (%). The gender was determined by microscopic analysis of gonad squashes at the end of the experiment (Guerrero and Shelton, 1974).

Study: Evaluation of potential action of phytochemicals on sex differentiation of tilapia by immersion treatments (OSU aquaculture laboratory).

Experiment 4: Immersion treatments in phytochemicals solution

A preliminary evaluation of the maximum tolerable

concentration (no-effect dose) of the chemicals daidzein, chrysin, caffeic acid and spironolactone was conducted. A series of concentrations on the range of 1 to 400 mg/l of each chemical was tested for each chemicals in a controlled temperature incubation system (27±1 °C), equipped with 1 l glass containers (24) and constant aeration, three replicates for each concentration with 60 all-male newly hatched tilapia larvae per container were exposed for 24 hr (overnight) and survival rate was recorded. For each chemical, the highest concentra-tion with zero mortality was determined as the no-effect dose; dimethyl sulfoxide (DMSO) was used as a solvent for all chemicals, given that it was the only common solvent for all used chemicals.

For the immersion experiment, mixed sex genotype tilapia larvae (Til-Tech USA) were placed in acrylic tanks (3.5 l) in a commercially designed water recirculation system (AHAB, Aquatic-Eco Inc®, Apopka, FL USA) at a density of 60 fish per tank, with three containers per treatment. The no-effect dose concentrations of the chemicals tested in the preliminary trial were as follows: daidzein (DAID) 400 mg/l, chrysin (CHR) 20 mg/l, caf-feic acid (CAFF) 40 mg/l, and spironolactone (SPIRO) 5 mg/l. A control group (CON) with the vehicle DMSO 1 ml/l, as well as androstatrienedione (ATD) 1.2 mg/l and 17α-methyltestosterone (MT) 400 µg/l were used; the last two were dissolved in a stock solution in metha-nol and re-dissolved in DMSO. Immersions took place 4 times at 10, 17, 21, and 28 days post-hatching; for this purpose fish were transferred to the controlled incuba-tion system mentioned above and kept in the chemical solution for 3 h at each immersion. During the experi-ment fish were fed with a semi-purified diet (CON) to avoid any possible exogenous sources of phytochemicals and steroids. The final sex was determined by micro-scopic analysis of gonad squashes at the end of experi-ment (Guerrero and Shelton, 1974) when fish attained a minimum total length of 3.0 cm.

Study: High pressure liquid chromatography (HPLC) determination of phytochemical concentration in fish tissue.

An analysis on the bioaccumulation of the 3 phyto-chemicals tested in the experiments using HPLC was conducted. In all cases an acid hydrolysis with 1M HCL in acidified methanol (100:5 v:v methanol:acetic acid) was used as extraction solution for concentration of phytochemicals from matrix (whole body tissue). The extraction procedure consisted of homogenization of individual fish in the extraction solution for 30 sec at 5000 rpm, later incubation at 37 ºC for 16 h (overnight); acidity was neutralized with 10N NaOH (equivalent volume to sample weight) to achieve a total dilution rate 1:10. Samples were either injected immediately to the HLPC or frozen at -80 ºC for further analysis. Recovery rate was estimated on the range of 95% for all three phy-


tochemicals. The HPLC system consist of a Beckman® 110B pump, 166 system gold detection module and a 406 system gold analog interface module; a Peaksimple® chromatography data system was used for chromato-gram analysis.

The detection by HPLC for chrysin was performed using a modified procedure from Shanhrzad and Bitsch, (1996). Mobile phase composition was 1M acetic acid in 80% methanol with a flow rate of 0.8 ml/min; wavelength for detection was 280 nm. A Synergi hydro 250X4.6 mm 4m (Phenomenex®) was used for the analysis. The detection limit was 50 ng/ml.

The detection by HPLC for daidzein was conducted us-ing a modified procedure from Hutabarat et al., (1998). Mobile phase composition was 33% acetonitrile in water: acetic acid (99:1, v:v) at a flow rate of 1.0 ml/min; wavelength for detection was 260 nm. A Synergi hydro 250X4.6 mm 4m (Phenomenex®) was used for the analy-sis. The detection limit was 45 ng/ml.

The detection by HPLC for caffeic acid was carried out using a modification of the method described by Walle et al., (1999). Mobile phase composition was water:ethylacetate:acetic acid (95.6:4.1:0.3 %) at a flow rate of 1.0 ml/min; wavelength for detection was 320 nm. An Ultrasphere ODS 150X4.6 mm 5m (Beckman®) was used for the analysis. The detection limit was 40 ng/ml.

Statistical Analysis

Mean final weight and survival were analyzed by one-way analysis of variance analysis, where significant dif-ferences were found, a Tukey test was performed (Zar, 1990), and for final sex ratio chi-square contingency tables were used. All statistical analysis was performed using the SAS version 8.02 (SAS Institute, Inc. Cary, NC USA).

RESULTS

Experiment 1No significant differences were observed on survival rate after 8 weeks, across all treatments (87.9±6.8 %) (Table 3). Growth rate results indicate a differential growth observed at the different points of evaluation (2, 4, 6, and 8 weeks) (Figure 1), being more evident at week 8 (Table 2) where in general CON showed a significant higher final mean weight (P0.05) compared to fish fed with the different phytochemicals. The final sex ratio of the experimental fish was not af-fected by the dietary administration of the tested phyto-chemicals; only the steroidal compounds ATD and MT achieved 50 and 100% masculinization rates, respectively when compared with the control treatment (Figure 2).

Experiment 2

Final mean weight (0.69±0.1 g) (Figure 3) and survival rate (97.7±2%) (Table 3) of all-male tilapia were not significant different across treatments after 6 weeks of feeding with the experimental diets. The final sex ratio was not altered from 100% genetically all-male due to the inclusion of CHR, DAID, CAFF, or SPIRO on the experimental diets across treatments.

Experiment 3Several setbacks were observed in this experiment. There was a very high variation in survival rates across treat-ments; 11.7 to 55% (Table 3). In two cases, for CHR and CAFF only one replicate could be evaluated towards the end of the experiment for sex ratio determination. On week 6 of the experiment a few significant differences in growth rate were established; however, towards the end of the experiment individual mean weight was not different among dietary treatments (Figure 4). SPIRO, MT and ATD treatments had a significant higher male percentage (P


DISCUSSION

This study addresses the possibility of using phyto-chemicals such as chrysin, daidzein, and caffeic acid to induce the required hormonal imbalance needed to observe an effect in sex differentiation in tilapia (Howell et al., 1994). Experimental conditions in vitro have shown that several phytochemicals block the biosynthesis and action of estrogens by (1) inhibition of aromatase activity and other steroid metabolism related enzymes, or (2) by competition for the estrogenic nuclear receptors (α and β ER), that could possibly mimic the sex-reversal effects of androgen treatments in fish (Collins et al., 1997, Le Bail et al., 1998, Jeong et al., 1999).

The first premise is based on the aromatase affinity for flavonoids. This enzyme is the only known enzyme able to catalyze the irreversible conversion of androstenione and testosterone into estrone and estradiol respectively; therefore aromatase is a good target for selective inhibi-tion because estrogen production is the last step in the biosynthetic sequence of steroid synthesis (Brodie et al., 1999). Given that the majority of information that sup-ports this theory is based on studies on in vitro condi-tions (Collins-Burrows et al., 2000, Le Bail et al., 1998, Jeong et al., 1999), when extrapolated to a live organism, the results obtained are diverse. Saarinen et al. (2001), showed that chrysin and 7-hydroxyflavone inhibit the formation of 3H-17α-estradiol from 3H-androstenione in human choriocarcinoma JEG-3 and human embryonic kidney HEK 293 cells, but when administered at a dos-age of 50 mg/kg to immature rats to evaluate the in vivo response, it failed to promote a reduced growth response in estrogen-dependent uterine enlargement. The lack of an in vivo response may be due to their relatively poor absorption and/or bioavailability and they state that the in vivo effects of flavonoids in aromatase inhibition cannot be predicted on the basis of in vitro results. In our study, given the lack of evidence of an active response on sex differentiation after phytochemical administra-tion by diet and immersion treatments, we can speculate that phytochemicals are not fully effective in affecting sex differentiation in tilapia.

The bioactivity of phytochemicals upon consumption is controversial. The evidence of high concentrations of phytochemicals in urine indicates that most of the chem-ical is eliminated by this means by humans and other vertebrates (Pelissero et al., 1996). In general, phyto-chemical absorption involves a series of conjugation and deconjugation steps facilitated by the gut bacterial flora and the liver. (Patisaul and Whitten 1999, Hollman and Arts 200, Miyahara et al., 2003). Differences in results between OSU and UJAT suggest two possibilities: First there is a chance that all these steps are not fully accom-plished given the degree of development of the digestive tract of the larval fish used for our experiments; second, there are possibilities of interspecies differences on the

degree on the efficiency of phytochemical absorption due to variation in the release of many different diges-tive enzymes by microflora involved in food metaboliza-tion processes (Bairagi et al., 2002) or specific rearing conditions that can influence digestive tract develop-ment and the specificity of the present bacterial flora in tilapia (LeaMaster et al., 1997). A possible indication of such a relationship between the degree of absorption and metabolization of phytochemicals and the degree of gut development and present microflora and their effect on sex differentiation could be the observed differences between Experiments 1 and 3 given the origin and rear-ing conditions. In Experiment 3, the chrysin final male percentage was higher (but not significant different) than the control group (Figure 5), and a similar occurrence was observed on the treatments spironolactone and androstatrienedione that had a significant effect on final sex ratio, contrary the results observed in Experiment 1, where only MT and ATD induce a sex inversion effect.

Thus, to establish to what extent larval fish absorb and metabolize phytochemicals could be an important factor to determine the possible in vivo bioactivity of flavonoids as endocrine disruptors. Future studies could focus on evaluating chrysin tissue concentrations of fish from the two locations, and contrast that if in both cases, the same trend as in Experiment 1 tissue concentrations of chrysin and daidzein is observed (Figures 7 and 8). For both phytochemicals, a diminution on tissue concen-tration throughout the feeding trial was observed in Experiments 1 and 3 in both genotypes (all-male and all-female), which could be related to an increased metabo-lization by enzymes produced by the digestive tract and microflora. Also the proper chemical identification of the free and sulfate-conjugated phytochemicals produced af-ter enzymatic reactions, which have a reported higher in vivo endocrine disruptor activity (Miyahara et al., 2003), is warranted due to a potential “activation” of some phytochemicals after metabolization that facilitates their absorption by intestinal membranes and transportation to the liver, re-metabolization, and delivery to target tis-sues (Kinjo et al., 2004).

Research on the bioavailability of flavonols, flavones, and flavanols has to be expanded. Attention must be given to the identification and quantification of their metabolites in body fluids and tissues, and sensitive and selective analytical methods will have to be developed (Hollman and Arts 2000). The HPLC detection meth-ods described in this provide preliminary information on the absorption of phytochemicals in fish. Although several modifications were made from the original papers that describe the analytical conditions, such as type of column, flow rate, and wavelength for detection, the chromatograms from Figures 9 and 10 confirm the presence of the parent compound in fish whole body tis-sue, although the free and conjugated metabolites need to be identified. For example, daidzein is metabolized


into dihydrodaizein, then equol, and finally O-demeth-ylangolensis (O-DMA) in humans. This has unknown estrogenic properties (Patisaul and Whitten, 1999). Once consumed, caffeic acid is transformed into a series of derivatives, mostly ferulic, isoferulic, and dihydroferu-lic acid that can be used as specific biomarkers for the bioavailability and metabolism of this phytochemical. However there is no information of the relevance of such derivatives as possible endocrine-disruptors chemicals (Rechner et al., 2001). Chrysin has apparent favorable membrane transport properties through cell membranes; however its absorption may still be seriously limited by a highly efficient conjugation metabolism by glucoroni-dation and sulfation by intestinal epithelial cells (Walle et al., 1999). Therefore, pharmacokinetic studies involved in the absorption of phytochemicals in fish need to be conducted.

Whether phytochemicals exert an anti-estrogenic effect once they bind to the steroid receptors is yet another point of controversy. It is well known that estrogen influ-ences the growth, development, behavior, and regula-tion of reproductive tissues in all vertebrates. Many of the effects of estrogens are mediated through binding to the estrogen receptors (ERs) (Cole and Robinson, 1992, McLachlan et al., 1992, Rickard and Thompson 1995, Matthews et al., 2000). Isoflavones such as daidzein bind to estrogen receptors with a lower affinity than 17α-es-tradiol (between 1 X 10-4 and 1 X 10-2 lower) on a molar basis. But still there are differences when it comes to different estrogen receptors sub-units given that bind-ing affinity can vary 5 to 20 times more efficiently to the ERα than the ERβ receptor (Messina et al., 2001). Chrysin binding affinity is 100,000 fold or more less than E2, and has very low or no agonistic activity in the ER’s (Kuiper et al., 1998). Ligand-binding specificities could influence the actions of phytochemicals as endocrine-disrupting agents at different target tissues (Thomas, 2000).

Another consideration when evaluating possible effects of phytochemicals on sex differentiation in juvenile fish is their effect on growth. In all feeding trials (Experi-ments 1, 2, and 3), no significant differences on final indi-vidual mean weight were observed on the phytochemi-cal fed fish after 6 or 8 weeks of feeding, when compared with the control group in all three experiments. No growth data was obtained during immersion experi-ments given the low carrying capacity of the biofiltration unit was limited the total amount of food provided per day (5%). Higher mortality rates were observed relative to Experiments 1 and 2 (Table 3). The suboptimal rearing conditions may have reduced the potency of treatments used (phytochemicals and steroidal compounds) or altering sex differentiation in tilapia.

A number of studies have focused on the use of alter-native steroidal compounds for sex inversion in fish. In this case, spironolactone was selected because of its

inhibitory effect on steroid synthesizing enzymes and androgen receptor affinity (Steelman et al., 1969, How-ell et al., 1994, Canosa and Ceballos 2001). Although spironolactone has promoted “paradoxical masculiniza-tion” effects in juvenile mosquito fish (Gambusia affinis), the responses observed were an expression of secondary male sex characteristics (Howell et al., 1994). The results observed in Experiment 4 (Figure 5) provide a promis-ing expectation for future research on the use of this steroidal compounds. Relative to the use of steroidal aromatase inhibitors such as ATD, distinct results have been reported, given that fadrozole (an steroidal aroma-tase inhibitor) can present estrogenic activity on the long run by competing with estrogen receptors and inducing an estrogen-like effect on DNA expression (Serallini and Moslemi 2001). Bertolla-Afonso et al., (2001) conducted another study with fadrozole that when administered on food for 15 or 30 days can influence sex determination in tilapia. The results indicate that treatment at dosages of 75 and 100 mg/kg achieve a 100% masculinization rate when fed for 30 days. However fadrozole is a more potent aromatase inhibitor than ATD, where a dosage of 150 mg/kg for 30 days only induces a 75% masculiniza-tion rate (Guiguen et al., 1999); in our study ATD only promoted a 50% masculinization effect in Experiment 1 (Figure 2), but a higher response of 97% in Experiment 4 (Figure 5), indicating a probable variability in genotype responses to steroidal aromatase inhibitors.

In conclusion, this study provides another resource in the evaluation of the effect in vivo of phytochemi-cals on sex differentiation in Nile tilapia. Although no significant effect on final masculinization percentage was observed using chrysin, daidzein, and caffeic acid, the validation of the presence of the parent compound with HPLC techniques in fish tissue encourages future research on the use of these chemicals for induced sex reversal. Also, the evidence supporting the possibility of alternative steroidal compounds, such as spironolactone, for sex inversion provides a future line of research.

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Ingredient (% / 100 g)Diet Formulation / 100 g

Control Chrysin(500 mg/kg)Spironolactone(500 mg/kg)

Daidzein(500 mg/kg)

Caffeic acid(500 mg/kg)

Casein 36 36 36 36 36Gelatin 6 6 6 6 6Dextrin 32.6 32.6 32.6 32.6 32.6CPSP 5 5 5 5 5Fish Oil 3.5 3.5 3.5 3.5 3.5Soybean oil 3.5 3.5 3.5 3.5 3.5Vitamin mix 4 4 4 4 4Mineral mix 4 4 4 4 4CMC 2 2 2 2 2L-Arginine 0.4 0.4 0.4 0.4 0.4L-Methionine 0.4 0.4 0.4 0.4 0.4L-Lysine 0.4 0.4 0.4 0.4 0.4Choline chloride 1 1 1 1 1Phospitan C 0.04 0.04 0.04 0.04 0.04Chrysin 0.05Spironolactone 0.05Daidzein 0.05Caffeic Acid 0.05Cellulose 1.16 1.11 1.11 1.11 1.11

Treatment (Diet)

Final Ind. Weight (g) by ExperimentAll-Male

OSU(Exp 1)

All-FemaleOSU

(Exp 2)

Mixed SexUJAT

(Exp 3)

Control 0.75±0.1 2.23±0.4a 0.10±0.07Chrysin 0.77±0.06 1.71±0.3 0.08±0.04Daidzein 0.67±0.1 1.91±0.4 0.10±0.06Caffeic Acid 0.64±0.1 1.70±0.2 0.11±0.04Spironolactone 0.63±0.1 1.38±0.5 0.07±0.1317α-Methyltestosterone 1.11±0.1b 0.11±0.051,4,6-androstatrien-3-17-dione 1.09±0.04b 0.09±0.06

Table 1. Diet formulation used for Experiments 1, 2, and 3. Values in percent of inclusion / 100g.

Table 2. Summary of mean final individual weight (± SD), for experiments 1, 2 and 3. Different letters indicate significant differences (P > 0.05) among groups by experiment.


Treatment

Survival % by ExperimentAll-Male

OSU(Exp 1)

All-FemaleOSU

(Exp 2)

Mixed SexUJAT

(Exp 3)

Mixed SexOSU

(Exp 4)

Control 97.6±2.0 88.3±5.0 55.5 47.2±12.0Chrysin 97.0±1.0 87.7±2.5 11.7 0.0±0.0Daidzein 98.6±1.5 88.9±5.3 30.2 25.0±1.7Caffeic Acid 97.6±2.3 77.8±10.5 11.7 47.7±7.0Spironolactone 97.6±1.5 85.0±1.6 46.0 31.6±23.317α-Methyltestosterone 92.8±2.5 28.4 37.2±21.81,4,6-androstatrien-3-17-dione 95.0±1.6 45.7 47.7±3.9

Figure 1. Mean weights (± std error) of all female tilapia (O. niloticus) by dietary treatment from Experiment 1, reared at the Aquaculture Laboratory of The Ohio State University. Control (CON), chrysin (CHR), daidzein (DAID), spironolactone (SPIRO), caffeic acid (CAFF), 17α-methyltestosterone (MT) and androstatrienedione (ATD).

0

0.5

1

1.5

2

2.5

3

0 2 4 6 8

CON

CHR

DAID

SPIRO

CAFF

MT

ATD

TIME (week)

WEIGH

T(g)

Table 3. Summary of final survival rates (% ± SD) for experiments 1, 2, 3 and 4.


Figure 2. Final percent of males (± std error) of all female tilapia (O. niloticus) by treatment from Experiment 1 at the Aquaculture Laboratory of The Ohio State University. Control (CON), chrysin (CHR), daidzein (DAID), spironolactone (SPIRO), caffeic acid (CAFF), 17α-methyltestosterone (MT) and androstatrienedione (ATD). ** indicates significant differences (P > 0.05) form CON group.

100

50.6

16.1

24.0

14.3

23.9

18.1

0

20

40

60

80

100

CON CHR DAID SPIRO CAFF MT ATD

**

**

Treatment

Males(%)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 2 4 6

TIME (week)

CON

CHR

DAID

SPIRO

CAFF

WEIGHT(g)

Figure 3. Final individual mean weights (± std error) of all male tilapia (O. niloticus) by dietary treatment from Experiment 2, reared at the Aquaculture Laboratory of The Ohio State University. Control (CON), chrysin (CHR), daidzein (DAID), spironolactone (SPIRO) and caffeic acid (CAFF).


0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

2 4 6 7TIME (week)

CONCHR

DAID

SPIRO

CAFF

MT

ATD

WEIGHT(g)

Figure 4. Final individual mean weights (± std error) of mixed sex tilapia (O. niloticus) by dietary treatment from Experiment 4, reared at the Aquaculture Laboratory of UJAT-Mexico. Control (CON), chrysin (CHR), daidzein (DAID), spironolactone (SPIRO), caffeic acid (CAFF), 17α-methyltestosterone (MT) and androstatrienedione (ATD)

84.4

100.0

63.6

96.4

55.073.0

59.0

0

20

40

60

80

100

CON CHR DAID SPIRO CAFF MT ATD

Treatment

**

****

Males(%)

Figure 5. Final percent of males (± std error) of mixed sex tilapia (O. niloticus) by treatment from Experiment 3 at the Aquaculture Laboratory UJAT-Mexico. Control (CON), chrysin (CHR), daidzein (DAID), spironolactone (SPIRO), caffeic acid (CAFF), 17α-methyltes-tosterone (MT) and 1,4,6- androstatrienedione (ATD). ** indicates significant differ-ences (P > 0.05) form CON group.


53.0

61.1

57.2

59.2

56.0

65.6

0

20

40

60

80

100

CON DAID SPIRO CAFF MT ATD

Treatment

Males(%)

Figure 6. Final male ratio (± std error) of mixed sex tilapia (O. niloticus) by treatment from Ex-periment 4 at the Aquaculture Laboratory of The Ohio State University. Control (CON), chrysin (CHR), daidzein (DAID), spironolactone (SPIRO), caffeic acid (CAFF), 17α-me-thyltestosterone (MT) and androstatrienedione (ATD).

19.2

9.9

0.8 0.3

5.9

2.1

0

10

20

30

40

50

2 4 6 8

Time (week)

All-female

All-male

ug/gFishTissue

Figure 7. Mean daidzein concentrations (± std error) in tilapia whole body from Experiment 1 (2, 4, 6, and 8 weeks) and Experiment 2 (4 and 6 weeks) reared at the Aquaculture Labora-tory of The Ohio State University. n=15 fish per week.


1.9

44.8

8.4

0.2

6.22.7

0

10

20

30

40

50

60

70

2 4 6 8

Time (week)

ug/gFishTissue

All-FemaleAll-Male

Figure 8. Mean chrysin concentrations (± std error) in tilapia whole body from Experiment 1 (2, 4, 6, and 8 weeks) and Experiment 2 (4 and 6 weeks) reared at the Aquaculture Labora-tory of The Ohio State University. n=15 fish per week.


Figure 9. Chrysin chromatograms of whole body tissue, a) Experiment 3 week 6 control group sample, b) Experiment 3 week 6 sample, c) 1.5 µg/ml std.


Figure 10. Daidzein chromatograms of whole body tissue, a) Experiment 3 week 4 control group sample, b) Experiment 3 week 6 sample, c) 1.5 µg/ml std.


Figure 11. Caffeic acid chromatograms of whole body tissue, a) Experiment 1 week 6 sample, b) Experiment 1 week 6 sample + spike (0.50 µg/ml caffeic acid), c) 1.17 µg/ml std

Cite as: [Author(s), 2005. Title.] In: J. Burright, C. Flemming, and H. Egna (Editors), Twenty-Second Annual Technical Report. Aquaculture CRSP, Oregon State University, Corvallis, Oregon, [pp. ___.]

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