LONG TERM TREATMENT WITH ESTRADIOL VALERATE ACTS AS ENDOCRINE DISRUPTOR IN ADULT GUPPY FEMALES (Poecilia reticulata Peters, 1859)

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1 Lucrări ştiinţifice Zootehnie şi Biotehnologii, vol. 39 (1) (2006), Timişoara LONG TERM TREATMENT WITH ESTRADIOL VALERATE ACTS AS ENDOCRINE DISRUPTOR IN ADULT GUPPY FEMALES (Poecilia reticulata Peters, 1859) MAG I. V.*, PETRESCU M. RUXANDRA**, FALKA I.* *Faculty of Animal Sciences and Biotechnologies, Cluj-Napoca, România **Faculty of European Studies, Cluj-Napoca, România TRATAREA PE TERMEN LUNG CU ESTRADIOL VALERAT ARE EFECT DISRUPTOR ENDOCRIN LA FEMELELE ADULTE DE GUPPY s(poecilia reticulata Peters, 1859) Numerous androgens and estrogens are used in sex reversal technology. Since the effect of estradiol valerate on embryos and newly born fry are useful, the effect of this hormone on adult females depends on term of treatment. We tested, here, the effect of treatment with estradiol valerate on adult guppy females. Our research wish to be useful especially to ornamental fish breeders, in YY males technology. Our results indicate an important role of the estrogens in parturitions regulation. Long-term treatment of adult females with estradiol valerate induces a premature parturition in guppy females wich are already in the gestation stage, but delay embrionary development of the following fertilized eggs. Key words: estradiol valerate, parturition, guppy. Introduction The Guppyfish. Poecilia reticulata, the popular and widespread guppyfish, has been brought for the first time in Europe in 1960 of Robert John Lechmere Guppy (BUD 2002, BUD ET AL. 2006). The species originates from South America: Venezuela, Barbados, Trinidad, Northern Brazil and Guyanas. It was widely introduced and established elsewhere, mainly for mosquito control. Several countries report adverse ecological impact after introduction. Africa: feral populations reported from the coastal reaches of Natal river from Durban southwards, as well as in the Kuruman Eye and Otjikoto Lake in Namibia. Europe: in countries having warm climate, or having thermal waters: Hungary, Romania (Băile Felix, Băile 1 Mai; MAG ET AL. 2006). Family: Poeciliidae (Poeciliids); subfamily: Poeciliinae; order: Cyprinodontiformes (rivulines, killifishes and live bearers); class: Actinopterygii (ray-finned fishes). They occur in warm springs, their effluents, weedy ditches and canals. Found in various habitats, ranging from highly turbid water in ponds, canals and ditches at low elevations to pristine mountain streams at high elevations. Has a wide salinity range but requires fairly 83

2 warm temperatures (23-24 C) and quiet vegetated water for survival. They feed on zooplankton, small insects, and detritus. Guppy is a benthopelagic, non-migratory, freshwater, brackish fish. ph range: 7 8; dh range: Climate: tropical, C; 14 N - 2 N, 67 W - 52 W. High minimum population doubling time, less than 15 months (tm= ; Fec= with multiple spawning per year). Female reaches 5 cm SL. Males mature at 2 months and females at 3 months of age ( Poeciliid fishes have a particular reproduction, based on an internal fecundation of the eggs. Embrionary development takes place into female s organism, being an ovoviviparous fish. Guppy males are smaller than guppy females and more colorful, the sexual dimorphism being observed at one-month age. The male s anal fin is turned into a copulatory organ (gonopodium) by modification of the third, fourth and fifth rays. This helps the male to introduce the sperm into the female s genital pore through a false and short accuplation, which makes the female to spawn for many times (even 6-7 times), every 4 s. After the accuplation, female stocks the sperms into receptaculum seminis for 5-6 months. Because of this particularity of reproduction, survival of a single female after unfavorable season can save the existence of the population. In this way, the nature substitutes the male when this is absent. Importance of Guppyfish. The guppyfish is a species of economical and commercial interest, having such an importance for the aquarium market, as also a model organism (physiology, endocrinology, carcinogenesis studies), in ecology (especially in behavioral ecology and sexual selection), ecotoxicology (as bioindicator), phylogeny (study of speciation phenomena, evolution of sex chromosomes in vertebrates, etc) and genetics research. Aquaristics is an agreeable and restful occupation, by which many of us are passionate. As many of us could permit this hobby, it must that some of us transform the hobby in profession and become biologic material producers, researchers or merchants. However much someone strives to render in numbers the world production of ornamental fishes, the statistics will be neither precise and nor concluding, and this is due to the fact that the small producers are much more numerous than the great producers and in the case of small and average animals there doesn t exist a precise evidence of yearly productions, such as exists in the case of great ones. In USA, the production in this industry of ornamental fish culture is one of many millions dollars. The yearly sales only in Florida are estimated to above 175 millions dollars to detail price (WATSON & SHIREMAN, 2005). The ornamental fish industry in Florida dates from the years 1930, but the great expansion was in the last 20 years. The farms from Florida produce now above 700 varieties of ornamental fishes and distribute them to en gross buyers and pet shops in whole world. Florida Agriculture Department has estimated these sales to 33.7 millions dollars on the year 1989 (WATSON & SHIREMAN, 2005). But, the main ornamental fish producers are the states of South-East Asia: Thailand, Singapore, Indonesia, Hong-Kong and Malaysia (WATSON & SHIREMAN, 2005). 84

3 The price of these fishes varies, decreasing from merchants to producers and function by the acquisition stock size. Figure 1. Summary of USA imports and exports of ornamental fish from 1990 to 1998 Table 1. Summary of top 10 freshwater ornamental fishes imported into the USA in 1992 (TAMARU & AKO 2000) Common name Scientific Name Percentage of total fish imported (1992) Poecilia reticulata Guppy Paracheirodon innesi Neon Tetra Xiphophorus maculates Platy Betta splendens Siamese Fighting Fish Carassius auratus Goldfish Gyrinocheilus aymonieri Chinese Algae-eater Poecilia sphenops Shortfinned Molly Paracheirodon axelrodi Cardinal Tetra Chanda lala Glassfish Capoeta tetrazona Tiger barb Total Number of individuals imported (1992) (x 10 6 ) X Although U.S.A. has an ornamental fish culture industry well developed, the imported quantities exceeding much above those exported. USDA Foreign Agricultural Service (in TAMARU & AKO, 2000) has ascertained a commercial deficit of about 34 millions dollars (Picture 1). About 75% of the freshwater fish 85

4 imported comes from South-East Asia. Here is the first 10 species top, the most required in 1992, between freshwater species, in the table 1 (top in which the guppyfish occupies detached the first position). Sex Reversal Technology. Many species of ornamental fish exhibit a marked sexual dimorphism due to the larger fins and more pigmented bodies, usually observed in males. This results in male fish commanding up to four times the price of females. Because of this price discrepancy, the culture of all-male stocks of aquarium fish has a significant economic advantage (PIFFERER & LIM 1997). Functional sex reversal from female to male is carried out by treatment with androgen hormones such as: 17-alpha-methyltestosterone, 19-norethynyltestosterone, 17-ethynyltestosterone, 9(11)-dimethyltestosterone, androstenedione methylandrosterone, 11-ketotestosterone, androsterone and testosterone undecanoate (YAMAZAKI 1983, MAG ET AL. 2004, MAG & BUD 2006). Another way in production of all male stocks is supermales (YY males) use in reproduction. The supermales can be obtained by male to female sex reversal, after estrogen treatment of XY embryos, and YY embryos respectively. For feminization, beta-estradiol, diethylstilbestrol, 17-alpha-ethynylestradiol, estrone and 3-benzoate estradiol are generally used. Research Purpose. In the ovoviviparous guppy, oocyte maturation is followed by intrafolicular fertilization and gestation. The fully developed embryos are ovulated at term just prior to parturition. Numerous androgens and estrogens are used in sex reversal technology. Estradiol valerate is at present tested by SABADOŞ, BUD and MAG (2006, unpublished data). Since the effect of estradiol valerate on embryos and newly born fry are useful, the effect of this hormone on adult females depends on term of treatment. We tested, here, the effect of treatment with estradiol valerate on adult guppy females. Our research wish to be useful, especially to ornamental fish breeders, in YY males (supermales) technology. Materials and Methods The hormone was orally administered to gravid females 5-24 days prior to the normal term of the parturition and approximately 6 s after parturition at doses of 0, 200, 300 and 400 mg/kg food respectively. Twenty guppy females for each lot were randomly chosen and bred into individual thanks. The number of parturitions in each lot was registered and presented in table 2. The hormone was given for 8 s consecutively (see table 2, underline bold text). Results and Discussions The number of parturitions that occurred in the control lot was constant, with a weak decreasing in the final part of the experiment, which is probably due to females long-term maintaining in the maternities. The lots 1, 2, and 3 respectively, registered a great decreasing in parturition number between the 4 th 86

5 of treatment and the 8 th (last) of treatment (see figure 2, table 2). This decreased number of parturitions is due to the fact that estradiol valerate induces early parturition at the begining of the treatment in guppies. After the end of estrogen treatment, the number of parturitions increased slowly. This indicates an important role of the estrogens in parturitions regulation. Long-term treatment of adult females with estradiol valerate induces a premature parturition in the guppy females which are already in the gestation stage, but delay embrionary development of the following fertilized eggs. Dissection of a few females that registered a delaying of the parturition revealed a high mortality of the embryos and their disrupted development. As females in the control lot presented almost always a round abdomen, the treated females presented a uncommon thinness of the abdomen and they lacked the dark spot of gestation. Table 2. Number of parturitions for each female lot at different doses of estradiol valerate/kg food Week of treatment Number of parturitions for each female lot at different doses of estradiol valerate/kg food 0 mg/kg (control) 200 mg/kg (lot 1) 300 mg/kg (lot 2) 400 mg/kg (lot 3) 1 st nd rd th th th th th th th th st 2nd 3rd 4th 5th 6th 7th 87 8th 9th 10th 11th control lot 1 lot 2 lot 3 Figure 2. Number of parturitions for each female lot at different doses of estradiol valerate/kg food

6 Conclusions Long-term treatment of adult guppy females with estradiol valerate induces a premature parturition in the females wich are already in the gestation stage, but delay embrionary development of the following fertilized eggs. In view of estradiol valerate use in sex reversal technology we recommend a short treatment of the adult females in the broodstock. Bibliography 1. BUD, I Acvaristica. Piscicultura ornamentală. Cluj-Napoca, Editura AcademicPres. 2. BUD, I. ET AL Acvaristica. Mică enciclopedie. Cluj-Napoca, Editura Risoprint. 3. MAG-MUREŞAN, I. V. ET AL The use of androgen hormones for artificial selection in Poecilia reticulata species. Buletinul Universităţii de Ştiinte Agricole şi Medicină Veterinară Cluj-Napoca. Seria zootehnie şi biotehnologii 60: MAG, I. V. ET AL Specii ornamentale de pesti resalbaticite in Lacul Peţea de la Baile 1 Mai. În volumul <Neobiota în România>, Cluj-Napoca, in press. 5. MAG, I. V., I. BUD 2006 Simultaneous treatment of both gravid females and their newly born fry with methyltestosterone gives the highest percentages of males in Poecilia reticulata (Peters, 1859). Simpozionul internaţional Realizări şi perspective europene în creşterea animalelor Iaşi, 4-5 mai PIFERRER, F., L. C. LIM 1997 Application of sex reversal technology in ornamental fish culture. Aquarium Sciences and Conservation 1(2): TAMARU, C. S., H. AKO Using Commercial Feeds for The Culture of Freshwater Ornamental Fishes in Hawaii. UJNR Technical Report, 28: WATSON, C. A., J. V. SHIREMAN 2005 Production of Ornamental Aquarium Fish. IFAS Extension YAMAZAKI, F Sex control and manipulation in fish. Aquaculture 33(1-4): TRATAREA PE TERMEN LUNG CU ESTRADIOL VALERAT ARE EFECT DISRUPTOR ENDOCRIN LA FEMELELE ADULTE DE GUPPY (Poecilia reticulata Peters, 1859) MAG I. V.*, PETRESCU M. RUXANDRA**, FALKA I.* *Facultatea de Zootehnie şi Biotechnologii, Cluj-Napoca, România **Facultatea de Studii Europene, Cluj-Napoca, România Rezumat: Numeroşi androgeni şi estrogeni sunt frecvent folosiţi în tehnologia inversiunii sexuale induse artificial. Pe când efectul estradiolului valerat asupra embrionilor şi alevinilor este util, efectul acestui hormon asupra femelelor adulte depinde de durata de aplicare a tratării. Ca urmare a acestui studiu, am abordat în prezenta lucrare efectul stradiolului valerat asupra femelelor adulte la guppy. Rezultatele noastre sunt utile în mod deosebit crescătorilor de peşti ornamentali care aplică în producţie tehnologia supermasculilor. Rezultatatele obţinute arată o importantă implicaţie a hormonilor estrogeni în reglarea funcţiei de reproducere şi în parturiţie. Tratarea pe termen lung a femelelor cu estradiol valerat induce o parturiţie prematură la femelele care se află într-un stadiu avansat al gestaţiei, dar are ca efect încetinirea şi întârzierea dezvoltării embrionare a următoarelor icre fertilizate. Cuvinte cheie: estradiol valerat, parturiţie, guppy. 88