Steroid androgen 17 alpha methyltestosterone used in fish farming induces biochemical alterations in zebrafish adults.

The 17 alpha methyltestosterone (MT) hormone is fed to Oreochromis niloticus larvae in fish farms with the purpose of inducing sex reversal. The aim of this study was to evaluate the toxicity and sub-lethality of MT (99.9% purity) and cMT (a commercial MT with 90% purity) in zebrafish (Danio rerio) adults, where the animals were exposed to concentrations of 0, 4, 23, 139, 833 and 5000 µg/L for 96 hours. Genotoxicity was evaluated by micronucleus test (MN), nuclear abnormalities (NA) and comet assay. A low genotoxic potential of MT was showed, inducing micronucleus, nuclear abnormalities and DNA damage in Danio rerio, depending on the use of MT or cMT, gender and tested concentrations. In the sub-lethality trials, there was a basal difference in the activity of the enzymatic biochemical markers for males and females, while the Glutatione S transferase (GST) activity decreased in all analyzed tissues, and for males the enzymatic activity decreased only in the intestine. Results suggest that MT has a toxic potential to fish because it alters enzymatic metabolic pathways and may pose a risk to the ecosystems.

Gonadal remodeling and hormonal regulation during sex change of juvenile dusky grouper Epinephelus marginatus (Teleostei, Serranidae), an endangered protogynous hermaphrodite fish.

Sex change was induced in Epinephelus marginatus juveniles using a nonsteroidal aromatase inhibitor (AI), a synthetic androgen (17α-methyltestosterone; MT), and a combination of both (MT + AI) in a 90-day experiment. A detailed remodeling of the gonads, the plasma level of gonadal steroids, and immunostaining of pituitary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and somatolactin (SL) cells were analyzed. Sex inversion reached the final spermatogenesis stages using MT, while AI triggered spermatogenesis, but reaching only the spermatid stage. Estradiol (E2) levels did not change in fish treated with AI but decreased throughout the experimental period in animals treated with MT and MT + AI. Testosterone (T) levels increased in animals treated with MT during the first 60 days (and combined with AI in the first 30 days), decreasing in all experimental groups at 90 days, while AI-treated animals had increased plasma 11-ketotestosterone (11-KT) levels after 90 days. In control fish, FSH- and SL-producing cells (ir-FSH and ir-SL) were restricted to pars intermedia (PI) of the adenohypophysis. Pituitary ir-FSH cells were decreased at the end of the experimental period in all treatments compared with the CT animals. LH-producing cells (ir-LH) were present in proximal pars distalis (PPD) and pars intermedia (PI) of adenohypophysis and did not change after the experimental period. The decreased number of ir-FSH cells at the end of the experiment in all treatments could be related to the negative feedback loop triggered by the increase in natural and/or synthetic androgens.

Complexes between methyltestosterone and ß-cyclodextrin for application in aquaculture production.

The inclusion complexes between 17-α-methyltestosterone (MT) and ß-cyclodextrin (bCD) were prepared and characterized in dissolution and solid phase. The complex promoted a sixfold increment in solubility of the hormone. It has a limited solubility and stoichiometry of 2:1 (bCD:MT) determined by DSC, NMR and solubility experiments, the association constant Ka=2846Lmol-1 and complex fraction of 76% (assessed by DOSY-NMR, in (1:3) DMSO/D2O). The association constant obtained in water by the solubility isotherms is 7540Lmol-1. 2D-ROESY experiments indicate the intermolecular orientation (complete inclusion of the hormone in the cavity). Simulations by molecular dynamics agreed with the formation of the inclusion complex 2:1. Release tests showed the slower release for the complexes, with 50% for lyophilization and 56% for malaxation. These results clearly demonstrate the complexation of MT in bCD, which formulations are promising for further applications involving this steroid in aquaculture, both for sexual reversal and in technologies of hormone in water sequestration.

Detection of a synthetic sex steroid in the American crocodile (Crocodylus acutus): Evidence for a novel environmental androgen.

Endocrine-disrupting contaminants (EDC's) are well known to alter sexual differentiation among vertebrates via estrogenic effects during development, particularly in organisms characterized by temperature-dependent sex determination. However, substances producing androgenic effects typically lack potency when tested in laboratory settings and are virtually unstudied in field settings. Here, we assay levels of a synthetic androgen, 17α-methyltestosterone (MT), in a heavily male-biased population of American crocodiles in the Tempisque River Basin of Costa Rica based on the recent hypothesis that this chemical is an EDC in developing crocodilian embryos. The presence of MT was documented in all field-collected samples of egg yolk and in plasma of all age classes in among population of crocodiles. Hatchlings exhibited higher plasma MT concentrations (102.1 ± 82.8 ng/mL) than juveniles (33.8 ± 51.5) and adults (25.9 ± 20.8 ng/mL). Among populations, crocodiles captured in the Tempisque River (62.9 ± 73.7 ng/mL) were higher in MT concentration than those from Tarcoles (13.3 ± 11.4 ng/mL) and negative controls (0.001 ± 0.0002 ng/mL). A mechanism for the bio-transport of MT and its subsequent effects is proposed.

Identification of neomales in South American catfish Rhamdia quelen on the basis of the sex ratio in the progeny

In the present study, we aimed to identify masculinized genotypic females of Rhamdia Quelen on the basis of the sex ratio of the progeny. Masculinizing diets containing 3 levels of 17α-methyltestosterone (MT; 60, 90, or 120 mg kg-1) were administered to R. quelenfry. Thereafter, 5 phenotypic males from each MT treatment were crossed with normal females, and their progenies were sexed. At least 1 male from each treatment group generated progeny with a higher female:male ratio. These males, designated M60, M90, and M120, were crossed again with 3 other normal females to re-evaluate the sex ratio of the progeny. Moreover, a normal male was used as a control. Again, we found a higher female:male ratio in the progeny of M60, M90, and M120. The control male produced 2sets of balanced progeny and 1 with a lower female:maleratio. Although these breeders did not generate all- female progeny (79.75 ± 7.20% of females), our findingsindicate that M60, M90, and M120 can be considered masculinized genotypic females of R. quelen, suggesting the involvement of autosomal genes or polygenic interactions in sex determination.

Identification of neomales in South American catfish Rhamdia quelen on the basis of the sex ratio in the progeny

In the present study, we aimed to identify masculinized genotypic females of Rhamdia Quelen on the basis of the sex ratio of the progeny. Masculinizing diets containing 3 levels of 17α-methyltestosterone (MT; 60, 90, or 120 mg kg-1) were administered to R. quelenfry. Thereafter, 5 phenotypic males from each MT treatment were crossed with normal females, and their progenies were sexed. At least 1 male from each treatment group generated progeny with a higher female:male ratio. These males, designated M60, M90, and M120, were crossed again with 3 other normal females to re-evaluate the sex ratio of the progeny. Moreover, a normal male was used as a control. Again, we found a higher female:male ratio in the progeny of M60, M90, and M120. The control male produced 2sets of balanced progeny and 1 with a lower female:maleratio. Although these breeders did not generate all- female progeny (79.75 ± 7.20% of females), our findingsindicate that M60, M90, and M120 can be considered masculinized genotypic females of R. quelen, suggesting the involvement of autosomal genes or polygenic interactions in sex determination. (AU)

Low dose of methyltestosterone in ovariectomised rats improves baroreflex sensitivity without geno- and cytotoxicity.

This study evaluated the effects of the isolated use of a low dose of methyltestosterone (MT) on cardiovascular reflexes and hormonal levels and its geno- and cytotoxic safety in ovariectomized rats. Female Wistar rats were divided into four groups (n = 6), respectively: SHAM (received vehicle methylcellulose 0.5%), SHAM + MT (received MT 0.05 mg/kg), OVX (received vehicle), and OVX + MT (received MT). Twenty-one days after ovariectomy, treatment was given orally daily for 28 days. The Bezold-Jarisch reflex (BJR) was analyzed by measuring the bradycardic and hypotensive responses elicited by phenylbiguanide (PBG) administration. The baroreflex sensitivity (BRS) was evaluated by phenylephrine and sodium nitroprussite. Myocyte hypertrophy was determined by morphometric analysis of H&E stained slides. Biochemical data were analyzed, as well as micronucleus assay. MT improved BRS and increased testosterone values, but did not change estradiol in the OVX group. MT did not promote changes in mean arterial pressure, heart rate, BJR, serum concentrations of troponin I, weight and histopathology of the heart. MT was able to restore the BRS in OVX rats. The geno- and cytotoxic safety of the MT was demonstrated by the absence of an increase in the micronucleus (PCEMN) or change in the ratio between normochromatic erythrocytes and polychromatic erythrocytes (NCE/PCE).

Somatic growth effects of intramuscular injection of growth hormone in androgen-treated juvenile Nile tilapia, Oreochromis niloticus (Perciformes: Cichlidae)

Little is known about the effects of the interaction of growth hormone (GH) with 17 a-methyltestosterone (17-MT) during fish growth. We evaluated this in the present study to assess the effect on fish growth. Fish in two batches of juvenile tilapia (Oreochromis niloticus) (approximately 5.0cm in length) were randomly assigned in triplicate to three treatments and a control group, distributed among 12 fiberglass tanks of 1 000L capacity (50 fish per tank) in an experiment covering a period of six weeks. The experimental groups were: a) fish treated with 17-MT and GH in mineral oil (RGH); b) fish treated with 17-MT and mineral oil without the addition of GH (R); c) fish treated with GH in mineral oil but not 17-MT (NGH); and d) fish of the control group, which were treated with mineral oil but not 17-MT or GH (N). The GH was injected into the fish at a rate of 0.625mg/g body weight. Morphometric data were recorded at the beginning of the experiment (T) and at 15, 30 and 45 days (T, T and T), and various indicators of growth were assessed: condition factor (K); survival percentage (S), feed conversion rate (FCR), percentage weight gain (WG) and (v) daily weight gain. The optimum dietary level was calculated assuming 5% food conversion to total weight in each group. During the experiment, the fish were provided with a commercial food containing 45% protein. The data showed that GH injection resulted in a greater weight gain in fish treated with 17-MT (the RGH treatment group), being particularly significant increase in weight during T and T (p<0.05). High values of K were found in the R and RGH treatments during the initial days of the experiment, which may have been a consequence of the better nutritional status affecting both weight gain and growth in body length, as a result of the additive effects of 17-MT and GH. The fish in groups not treated with 17-MT and treated with 17-MT and added GH showed greater increases in WG per day, higher K values and lower FCRs than fish in the other groups, which suggests that greater feed efficiency occurred in the hormone-treated fish. Fish in the RGH treatment showed the most growth, suggesting a possible interaction between 17-MT and injected GH.

Actualmente, durante el crecimiento de los peces existe poco conocimiento sobre los efectos de la interacción de la hormona del crecimiento (HC) con 17 α-metiltestosterona (17-MT). En el presente estudio los peces en dos lotes de tilapia (Oreochromis niloticus) (5.0cm de longitud), fueron asignados al azar por triplicado a tres tratamientos y un grupo control, distribuidos en 12 tanques de fibra de vidrio de 1 000 litros (50 peces por tanque), en un período de seis semanas. Los tratamientos fueron: a) peces tratados con 17-MT+HC en aceite mineral (RGH), b) peces tratados con 17-MT+aceite mineral sin la adición de HC (R), c) los peces que no fueron tratados con 17-MT-tratado+HC en aceite mineral (NGH), y d) los peces que no fueron tratados con 17-MT+aceite mineral (N). La hormona de crecimiento humano recombinante (Humatrope, Eli Lilly & Co., Windlesham, Inglaterra), se inyectó en el pez con una dosis de 0.625mg por gramo de peso corporal. Los datos morfométricos se registraron al comienzo del experimento (T) y en los días 15, 30 y 45 (T, T y T), Se registraron diversos indicadores de crecimiento: factor de condición (K), porcentaje de supervivencia (S), la tasa de conversión alimenticia (FCR), porcentaje de ganancia de peso (GP) y el aumento de peso al día. El nivel óptimo dietético fue calculado suponiendo 5% de conversión de alimentos al peso total de cada grupo. Durante el experimento fue usada una dieta comercial con el 45% de proteína. De los resultados presentados, es evidente que la inyección de HC dio lugar a una mayor ganancia de peso en el 17-MT-los peces tratados (el grupo de tratamiento RGH), y la diferencia fue significativa, tanto en T y T (p<0.05) para ambas comparaciones. De manera similar, los altos valores de K se presentaron en los tratamientos R y RGH durante los primeros días de cultivo. Esto puede haber sido asociado con un mejor estado nutricional que afectó tanto el desarrollo de peso y la longitud del cuerpo del pez, como resultado del efecto aditivo de 17-MT y GH. Los tratamientos no andrógenos y los grupos tratados con andrógenos y con HC mostraron un mayor incremento en la ganancia de peso por día, los mayores valores de K y menores tasas de conversión del alimento, lo que sugiere una mayor eficiencia de la alimentación en los peces tratados con hormonas. Peces en el tratamiento RGH mostraron el mayor crecimiento, lo que sugiere una posible interacción entre el 17 de α-metiltestosterona (17-MT) y hormona de crecimiento inyectada.

Salinidade da água e suplementação alimentar com microalga marinha no crescimento e masculinização de tilápia do Nilo / Water salinity and food supplementation with marine microalgae on growth and masculinization of nile Tilapia

O objetivo deste trabalho foi avaliar a influência da salinidade da água e suplementação alimentar com Spirulina platensis no crescimento e masculinização de Oreochromis niloticus, tilápia do Nilo. Na primeira fase, além da S. platensis, foi ofertada ração microparticulada (50% PB; 4500 Kcal/kg) contendo o andrógeno 17-α- metiltestosterona. Na segunda etapa, as tilápias foram cultivadas por mais 32 dias em água doce, alimentadas apenas com ração (38% PB; 1900 Kcal/kg). Os delineamentos foram divididos em três tratamentos com cinco repetições cada. Os animais (peso inicial: 0,01 ± 0,02 g, comprimento inicial: 1,0 ± 0,03 cm) ao final de 28 dias apresentaram crescimento médio em peso e crescimento total em comprimento de 1,64 ± 0,10 g e 4,84 ± 0,24 cm; 1,59 ± 0,18 g e 4,94 ± 0,12 cm e 1,58 ± 0,09 g e 4,84 ± 0,02 cm para as salinidades 0, 15 e 25 g.L-1, respectivamente. A masculinização mais satisfatória foi alcançada pelos peixes cultivados a 15 g.L-1, ao qual obtiveram 90% de machos. A análise de variância não evidenciou diferenças significativas (p>0,05) entre os tratamentos para o desenvolvimento em ambas as fases de cultivo e para a taxa de masculinização ao final do experimento. As salinidades testadas não interferiram no desempenho dos peixes, propondo que a microalga S. platensis pode ser utilizada com êxito como suplemento alimentar para tilápias do Nilo nas condições testadas.

The aim of this study was evaluate the influence of water salinity and diet supplemented with Spirulina platensis on growth and masculinization of Nile tilapia. In the first phase, in addition to S. platensis, was offered microparticulate diet (50% CP, 4500 Kcal/kg) containing 17-α-methyltestosterone androgen. In the second phase, the tilapias were cultivated for 32 days in fresh water, fed only with ration (38% CP, 1900 kcal/kg). The designs were divided into three treatments with five replicates each. The animals (initial weight: 0,01 ± 0,02 g, initial length: 1,0 ± 0,03 cm) after 28 days showed average weight growth and total length growth of 1,64 ± 0,10 g; 4,84 ± 0,24 cm, 1,59 ± 0,18 g; 4,94 ± 0,12 cm, 1,58 ± 0,09 g; 4,84 ± 0,02 cm, in salinities 0, 15 and 25 g.L-1, respectively. The indices of improvement sexual inducing were achieved by animals cultured at 15 g.L-1, which reached 90% of masculinization. Analysis of variance showed no significant differences (p>0,05) among treatments for development in cultivation phases both and the masculinization rate in the experiment final. The salinities tested did not interfere with the performance of fish, suggesting that the microalgae S. platensis can be successfully used as a food supplement for Nile tilapia in the tested conditions.

Growth and survival of tilapia Oreochromis niloticus (Linnaeus, 1758) submitted to different temperatures during the process of sex reversal / Crescimento e sobrevivência de tilápias Oreochromis niloticus (Linnaeus, 1758) submetidas a diferentes temperaturas durante o processo de inversão sexual

The objective of this research was to evaluate the sex reversal technique using 17α--methyltestosterone (MT) hormone, submitted to temperature modification of fry Nilo tilapia storage, aiming to get the data of sex reversal combined with growth performance and fry survival. The experiment was performed at UFLA Fish Culture Station, using tilapia fry (0,008 ± 0.002 g e 0,9 ± 0.1 cm) obeying a totally randomized experimental delineation in a factorial scheme 4x4, in 4 temperatures (26º, 28º, 30º, 32ºC) and 4 hormonal doses (0, 20, 40, 60mg of MT/kg of ration) during 28 days, with 5 repetitions. As temperature raised, weight gain rate, size and survival increased (p<0.01); however, this temperature raise was not effective in modifying males ratio (p>0.01), which occurred only due to the used hormone treatment. The dose of 40 mg of MT/kg of ration provided similar results to those of 60mg of MT/kg of ration. Hence, the temperature band from 26º to 32ºC does not affect sex reversal rate, but temperatures around 30ºC improves the performance of tilapias related to the growth and survival, and the dose of 40 mg of MT/kg of ration is enough to achieve monosex populations.

Objetivou-se, neste trabalho, avaliar a técnica de inversão sexual utilizando hormônio 17α-metiltestosterona (MT), submetidas à modificação da temperatura de estocagem das pós-larvas de tilápia, visando obter os melhores dados de inversão sexual aliado à performance de crescimento e sobrevivência das pós-larvas. O experimento foi conduzido na Estação de Piscicultura da UFLA, utilizando pós-larvas (0,008 ± 0,002 g e 0,9 ± 0,1 cm) de tilápia em delineamento experimental inteiramente casualizado em esquema fatorial 4x4, com 4 temperaturas (26º, 28º, 30º e 32ºC) e 4 doses hormonais (0, 20, 40 e 60mg de MT/kg de ração) durante 28 dias, com 5 repetições. À medida que se elevou a temperatura, a taxa de ganho de peso, o tamanho e a sobrevivência foram maiores (p<0,01); entretanto, esse aumento na temperatura não foi suficiente para alterar a proporção de machos (p>0,01), que ocorreu apenas em função do hormônio utilizado. A dose de 40 mg de MT/kg de ração proporcionou resultados semelhantes aos da dose de 60 mg de MT/kg de ração. Portanto, a faixa de temperatura entre 26º e 32ºC não influencia na taxa de inversão sexual, mas temperaturas em torno de 30ºC melhoram o desempenho das tilápias quanto ao crescimento e à sobrevivência, e a dose de 40 mg de MT/kg de ração é suficiente para a obtenção de populações monossexo.