Oral Administration of Testosterone in Fish Diet Affect Sex Differentiation and Testis Development in Tilapia.

Transcription

1 Research Journal of Agriculture and Biological Sciences, 6(6): , , INSInet Publication Oral Administration of Testosterone in Fish Diet Affect Sex Differentiation and Testis Development in Tilapia. 1 Wahbi, O.M., 2 Shalaby, S.H. 1 Fish Reproduction lab 2 Fish Nutrition Lab National Institute of Oceanography and Fisheries, Alexandria, Egypt. Abstract: Fries of Tilapia Zillii, Tilapia Nilotica and Red Tilapia were fed hormone-free (control diet) and hormone treated diets of 30% crude protein. Methyl-testosterone (Mt) was incorporated in the diets at 3 levels (60,120 and 200 mg Mt / kg feed ) and administered to 7 days post hatching fries for a period of 28 days. Male's ratios after 30 days post-hormone treatment substantially differ from control % males were produced with low dose of 60 mg Mt / kg feed. Higher dosage levels of 120 and 200 mg Mt / kg feed had an inhibitory effect on oogenesis, the degree of inhibition was dependent on the level of androgen and fish species. Remarkable propagation of somatic element in gonads occurred and a high % of neutral gonad was noticed at hormonal level of 120 and 200 mg Mt / kg feed. Plasma testosterone level decreased significantly in six moths' old sex inversed males (fed diet of 60mg Mt / kg feed ) compared to control specially in Tilapia Nilotica and Tilapia Zillii. Histological characteristic of mature sex reversed males show spermatogenic cysts with cells in early stages of development were predominating while in control testes cysts with predominant spermatozoa were seen. Key words: Hormone treated diet, sex reverse, histology, testosterone level, Tilapia. INTRODUCTION Artificial sex reversal using sex steroids has been accomplished in several fish [15,26,9,10] performed sexinversion in various tilapia species. 17α-methyltestosterone was the most common applied androgen tested in Cichlidae [5,2]. Yamamoto [31] reported the labile period in which gonads are highly sensitive to the action of exogenously administered steroid in Cichlids is the period in which differentiation occurs, just following hatching and lasts for a short period of days. Pandian & Sheela [23] obtained a complete sex reversal in which the phenotypic sex is reversed producing viable fertile fish when the hormone is applied at beginning of the sexually undifferentiated stage and continued through gonadal development. The late administration namely, after onset of sex differentiation leads to failure to achieve sex reversal [36]. Excessive doses of hormone lead to sterility or paradoxical feminization following aromatization of androgens to estrogens [8], although sub-optimal treatments resulted in intersexes [24]. Several histological studies have been done on sex differentiation on fish belonging to the genus tilapia [18,21,35] days after hatching gonads begin to differentiate to % and & [20]. Whereas the germ cells entered into meiosis in the ovary and steroid producing cells increases in number 35 days after hatching [19]. Active spermatogenesis began in testis by 100 days after hatching; however a rapid increase in leydig cells occurred in testis of fish after 70 days following hatching [20]. Rothbard et al., [25] examined the changes in steroid concentrations during sexual ontogenesis in 3 species of tilapia including Oreochromis niloticus, they found that period between 3-5 weeks, corresponds to the critical phase in sex reversal on treatment with exogenous androgens, endogenous steroid during this phase are very low. In 5-10 weeks, endogenous steroids increase gradually. They presumed that testosterone production is a result, rather than the cause of sex differentiation. Nakamura and Nagahama, [20] detected low testosterone level in % s at days after hatching, just after initial sex differentiation. The present attempt to manipulate the sex in tilapia through feeding 17α- Methyl-testosterone at doses ranging from ( mg Mt / kg feed diet) for 28 days affect the sex differentiation and induces production of functional males. Corresponding Author: Wahbi, O.M., Fish Reproduction lab, National Institute of Oceanography and Fisheries, Alexandria, Egypt. 946

2 MATERIALS AND METHODS Newly hatched fries of Tilapia Zillii, Tilapia Nilotica and Red Tilapia (T.Z, T.N and R.T) were counted and stocked in tanks of capacity (36 L), in densities of 50 ± 5 fry / tank. Water was kept at a constant temperature of 28± 0.5 Cº under natural light condition. Four treatment feeds were prepared using the same feed composition at 30% crude protein. Ingredients composition of the control diet is presented in Table(1). One treatment feed served as control and the 3 others were formulated from control diet to contain 17αmethyltestosterone (Mt, Sigma chemicals Co, USA) in ratios of 60, 120 and 200 mg Mt / kg feed prepared by the alcohol preparation method [9]. The hormones treated diet were prepared fresh every week. The various hormone levels were selected based on experiments with tilapia [18,26]. Each diet was fed to duplicate aquaria. The feed amounts were adjusted at 8,6 and 4% of the body weight for the 1-8,9-16 and week, respectively. The feed amount was given two times a day in equal proportions, for 6 consecutive days weekly. Fish were fed the diet supplemented with 17αmethyltestosterone for duration of 28 days started from 7 th day post hatching. Then they were further reared on the control diet until sex ratios could be determined through histological and macroscopic observations (30 day post hormonal treatment). For histological observations fries were collected randomly from each experimental and control fish groups and sexed using light microscope. Fish segment were fixed in Bowin's fluid then dehydrated and embedded in wax (m.p.56cº), sectioned at 4µm thickness and stained with Hematoxylin &Eosin. Sex inversed males that feed diet supplemented with (Mt) in ratio of 60 mg Mt / kg feed from all treated groups and control were left to grow till maturation (6months) and the histological characteristics of their testis were determined. At the end of experiment, blood was pooled from fish after severing caudal peduncle and plasma were collected individually for hormonal study. The plasma testosterone level of sex inversed and control 6 month's old males were measured by radioimmunoassay, in order to clarify the problem of a possible hazard in fish previously exposed to androgen. T- test was used to evaluate the significant changes and the differences were accepted at 5% level. RESULTS AND DISCUSSION Oral administration of (Mt) in diets of 7 days old tilapia fries up to 28 days is shown in Table (2). Control diet (0%Mt) comes closest to the 1:1% / & ratio with % sex ratio range from %. Methyltestosterone caused sex reversal at dosage level of 60mg Mt / kg feed with ratio ranging from % in different tilapia species. Independent of the dose the percentage of % s is higher in the treated groups. At higher doses of treatment (120,200mg Mt/kg feed) the masculinizing effect of (Mt) is pronounced, furthermore intersexes individuals (indistinguishable sexed gonads) are identified and their number increase proportional to hormone level. Histological Examination: Histological examination revealed no differences in gonadal tissues between control % s and treated ones. Testicular tissues aren't observed in the developed ovaries of treated fish groups. However, ovaries of some fish in each of these groups are retarded. These changes are consistent throughout the entire gonad and aren't restricted to isolated foci. Methyltestosterone suppressed oogenesis.this inhibitory effect on the development of oocytes is dependent on the dose of (Mt). Control ovary contains many oocytes at peri-nucleolus stage Figs (1, 6 &10). In treated Tilapia Zillii, ovary has decreased number of oocytes. The oocytes were scattered in gonad stroma with somatic cells in between Figs (2, 3 &4) or arranged around the ovarian cavity as in Tilapia Nilotica Figs (7& 8 )and Red Tilapia Fig (11). At dosage of 120, 200 mg Mt / kg feed, the hormone caused a remarkable propagation of somatic elements of gonad. Somatic elements are located in all ovary and few germ cells either at margin of ovarian cavity or solitary in somatic tissue, Figs (5, 9 &12). These two effects on germinal and somatic elements are seen in the same ovary leading to neutral gonads in all species of tilapia. Table1: Ingredients composition of control diet (30% crude protein) Ingredients Control diet Fish meal 35 Soybean meal 28 Yellow corn 17 Corn starch 12 Sunflower oil 6 Vitamin mix 1 & Mineral mix 2 2 Total 100 Proximate analysis (% DM) Dry matter 95.4 Crude Protein(CP) 30 Ether extract 11 Crude fiber (CF) 4 Nitrogen free extract (NFE) 44.5 Ash Vitamin mixture (g/100g) was IU, IU, 0.8g, 80 Mg, 0.32g, 0.12g, 0.8g, 0.8mg, 1.6g, 80mg, 4mg, 40mg,of vitamin A, D3, E, K, B1, B2, B6, Pantothenic acid, B12, Niacin, Folic acid Biotin, Choline chloride,respectively. 2. Mineral mixture (g/100g) was 12.75, 72.85, 0.55, 0.25, 0.02, 5, 2.5, 0.08, 0.05,0.01 and 6mg of MgSO 4.7H 2 O, CaHPO 4.2H 2 O, ZnSO 4.7H 2 O, MnSO 4.4H 2 O, Cal 2.O 6. H 2 O, KCl, FeSO 4.7H 2 O,CuSO 4.5H 2 O,CoSO 4.7H 2 O,CrC 3.6H 2 O and NaCL, respectively. 947

3 Fig. 1: Control T.Z. ovary. ovarian cavity (Oc). Fig. 5: Neutral gonad of fish treated with 120 and 200 mg Mt/Kg feed. germ cell (Gc). Fig. 2: Ovary of female fish treated with 60 mg Mt/Kg feed. oocyte (Arrows). Fig. 6: Control T.N. ovary. Fig. 3: Ovary of female fish treated with 120 mg Mt/Kg feed. oocyte (Arrows). Fig. 7: Ovary of female fish treated with 60 mg Mt/Kg feed. ovarian cavity (Oc). Fig. 4: Ovary of female fish treated with 200 mg Mt/Kg feed. oocyte (Arrows). Microscopic examination of testis of control male after 6 months reveals predominant spermatozoa Figs (13, 15 &17), while in sex reversed male fed diet containing 60 mg Mt / kg feed, cysts with Fig. 8: Ovary of female fish treated with 120 mg Mt/Kg feed. ovarian cavity (Oc). 948

4 Fig. 9: Neutral gonad of fish treated with 120 and 200 mg Mt/Kg feed. ovarian cavity (Oc), germ cells (Gc). Fig. 12: Neutral gonad of fish treated with 120 and 200 mg Mt/Kg feed. ovarian cavity (Oc), germ cells(gc). Fig. 10 : Control R.T. ovary. Fig. 13: Control T. Z testis, showing spermatozoa in lobule. Fig. 11: Ovary of female fish treated with 60 mg Mt/Kg feed. ovarian cavity (Oc), sertoli cells (Sc). spermatogenic cells in early stages of development are predominating and distribute throughout the entire gonad Figs (14, 16 &18). Treated males of T.Z that fed diet containing 60 mg Mt / kg feed showed testis to great extant as control one. Fig. 14: Treated T. Z testis, showing few spermatozoa in lobule Hormonal Study: Testosterone level in plasma of mature sex reversed T.N.and R.T.is significantly decreased having values of 37.64±5.9 and 45.29±3.9 ng/ml, while in the sexually active % s 41.18± 4.5 and 49.10± 4.1ng/ml respectively, Table (3). An insignificant decrease in testosterone level is detected in the % s of T.Z ±4.3 compared to ±3.8 ng/ml in control ones. 949

5 Table 2: Sex ratios of fries 30 days post hormonal treatment Fries species Hormone conc.mg/kg No. of fries sexed Male % Neutral sex % T.Z. O T.N R.T Table 3: Testosterone level in 6-months control and sex reversed males. Experimented groups Testosterone level ng/ ml - T.Z T.N R.T Control males ± ± ± Treated males 12.78± ±5.9* 45.29±3.9* *Significant at P>0.05 Fig. 15: Control T. N testis, showing spermatozoa in center of lobule. Fig. 17: Control R.T testis, showing great number of spermatozoa. Fig. 18: Treated R.T testis with predominant early stages of sperm development Fig. 16: Treated T. N testis, showing pridomant of sperm developmental stages. 1, primary spermatogonia; 2, secondary spermatogonia; 3, primary spermatocyte; 4, secondary spermatocyte; 5, spermatide; 6, spermatozoa; 7, Sertoli cell; 8, leydig cell. 950

6 Discussion: Ovarian and testicular differentiation in tilapia began by the stages of 15 and 30 days after hatching respectively [21]. In present investigation, hormonal treatment was carried out 7-35 days after hatching. As a result of hormonal treatment with methyl-testosterone, ovaries showed a definite tendency toward involution similar to that described in T. aurea by Yamamoto [31]. He reported that (Mt) caused the elimination of most of the germinal and stromal cells in gonads. Also similar result was reported by Nolan et al., [22] and Swanson et al., [27] in roach. On the other hand,(mt) had inhibitory effect on the development of reproductive elements of gonads, consequently, ovary was almost occupied with somatic elements. This stimulatory effect on somatic elements was also reported by Wolf et al., [30] and Fredrick et al., [6], upon treatment with testosterone gonads were composed mainly of connective tissue with only occasional germ cells. Sex ratio in the treatment receiving diet (60 mg Mt /kg feed ), showed the highest sex reversal. This diet falls within the effective concentration for Tilapia 40-70mg Mt / kg feed [4], for goldfish 125 ppm [32] and zebra fish ppm [33]. In this work, the treatment of tilapia with 17α-methyl-testosterone influenced the sex ratio in favor of % s, also when higher dosages of methyl-testosterone were administered (120 and 200 mg Mt /kg feed) females showed retardation.these findings were also recorded by William et al., [29] ; Macintosh et al., [16] and Tayamen & Shelton [28]. The excess of % s over & s in tilapia treated with 120, 200 mg Mt /kg feed was similar to the masculinizing effect observed in catfish Heteropneustes fossilis immersed in concentration of 100 ug/l Mt [14] and Chinook salmon immersed in 200 ug/l Mt [1], as well as treatment with(mt)at concentration of 400 ug/l produced a maximum of 55% masculinization in all & Amago salmon [17]. The decrease in plasma testosterone level recorded in T.N and R.T in present work in sex inversed % s was previously found in tilapia [3] ; in seabass [11-13] and in anemonefish by Godwin & Thomas [7]. Also the insignificant difference between level of testosterone in control and sex-reversing T.Z at maturation in this work was also found in sparidae by Yeung and Chan [34]. They reported that low level of testosterone in the sex-inversed fries may be correlated to the lack of sexual behavior in such fish which in tern delay maturation of testis in treated males. The general conclusion which can be drawn is that administration of testosterone in diet of tilapia fries for period of 28 days from 7 th day post hatching caused male production at level of 60mg Mt/kg diet, at higher levels of mg Mt/kg diet it caused ovarian retardation and production of neutral gonads. Testosterone level in mature males decreased and testis structure is altered. REFERENCES 1. Baker, I.J., I.I. Solar and E.M. Donaldson, Masculinization of chinook salmon (Oncorhynchus tshawytscha) by immersion treatments using 17αmethyltestosterone around the time of hatching. Aqua., 72: Beardmore, J.A., G.C. Mair and R.I. Lewis, Monosex male production in finfish as examplified by tilapia,applications, problems and prospects. Aqua., 197: Bhandari, R.K., M. Nakamura, T. Kobayashi and Y. Nagahama, Suppression of steroidogenic enzyme expression during androgen induced sex reversal in Nile tilapia (Oreochromis niloticus).gen.comp. Endocrinol., 150(3): Clemens, H.P. and T. Inslee, The production of unisexual broods of Tilapia mossambica sex -reversed with methyltestosterone. Trans. Amer. Fish.Soc., 97(1): Desprez, D., E. Géraz, M. Claude, C. Mélard, P. Bose and J.F. Baroiller, Production of a high percentage of male offspring with a natural androgen, 11 β - hydroxyandrostenedione (11 βoha 4 ), in florida red tilapia. Aqua., 216(1-4): Fredrick, W., Goetz, M. Edward, Donaldson; A. George, Hunterand Helen, M. De., Effects of estradiol-17 β and 17α-methyltestosterone on gonadal differentiation in the coho salmon, (Oncorhynchus kitsuch). Aqua., 17(4): Godwin, JR. and P. Thomas, Sex change and steroid profiles in the Protandrous anemonefish, Amphifrion melanopus (Pemacentridae, Teleostei). Gen. Comp. Endocrinol., 91: Goudie, C.A., B.D. Redner, B.A. Simco and K.B. Davis, Feminization of Channel catfish by oral administration of steroid sex hormones. Trans. Am. Fish. Soc., 112: Guerrero, R.D., Use of androgens for production of all male Tilapia aurea steindachner). Trans. Am. Fish. Soc., 104: Guerrero, R.D., Tilapia mossambica and Tilapia zillii treated with ethyltestosterone for sexreversal. Kalikasan,Philipp.J. Biol., 5: Guiguen, Y., B. Yalabert, E. Thouard and AT. Fostier, Changes in plasma and gonadal steroid hormones in relation to the reproductive cycle and the sex inversion process in the protandrous seabass, Lates calcarifer. Gen. Comp. Endocrinol., 92(3):

7 12. Guiguen, Y., B. Yalabert, A. Benett and AT. Fostier, Gonadal in vitro androstenedione metabolism and changes in some plasma and gonadal steroid hormones during sex inversion of the protandrous seabass, Lates calcarifer. Gen. Comp. Endocrinol., 100(1): Guiguen, Y., JF. Baroiller, MJ. Ricordel, k. Iseki, O.M. McMeel, S.A. Martin and A.T. Fostier, Involvement of androgens in the process of sex differentiation in two fish species: rainbow trout (Oncorhynchus mykiss )and tilapia (Oreochromis niloticus). Mol. Reprod. Dev., 54(2): Haniffa, M.A., S. Sridhar and M. Nagarajan, Hormonal manipulation of sex in stinging catfish Heteropneustes fossilis (Bloch). Current Science, 86(7): Hunter, G.A. and E.M. Donaldson, Hormonal sex control and its application to fish culture. In Fish Physiology, IX: Hoar, W.S. and E.M. Donaldson, Academicpress, NewYork. J. Macintosh, T. Vaghese and G. Satyanarayana, Hormonal sex reversal of wild spawned tilapia in India. J. Fish. Biol., 26: Nakamura, M., Study of susceptibility of sex reversal after a single two hour treatment of androgen in Amago salmon. Fish. Sci., 60: Nakamura, M. and M. Iwahashi, Studies on the pratical masculinization in tilapia nilotica by the oral administration of androgen. Bull. Jap. Soc. Sci., Fish., 486: Nakamura, M. and Y. Nagahama, Steroid producing cells during ovarian differentiation of tilapia,sarotherodon niloticus. Dev.Growth Differ. 27: Nakamura, M. and Y. Nagahama, Differentiation and development of leydig cells and changes of testosterone levels during testicular differentiation in tilapia Oreochromis niloticus.fish Physiology and Biochemistry, 7(1-4): Nakamura, M., H. Takahashi and M. Iwahashi, Gonads sex differentiation in tilapia nilotica Oreochromis niloticus. J. Exp. Zool., l.281(1-4): Nolan, M., S. Jobling, G. Brighty, D. Sumpter and C.R. Tyler, A histological description of intersexuality in the roach. J.Fish Biol., 58(1): Pandian, T.J. and S.G. Sheela, Hormonal induction of sex reversal in Fish.Aqua.,138: Popma, T.J. and B.W. Green, Sex reversal of tilapia in earthen ponds. Research and Development Series. International Center for Aquaculture, Alabama Agricultural Experiment station, Auburn University Auburn, AL., p: Rothbard, S., B. Moav and Z. Yaron, Changes in steroid concentrations during sexual ontogenesis in tilapia. Aqua., 61: Shelton, W.L., D.K. Hopkins and L.G. Jensen, Use of hormones to produce monosex tilapia for aquaculture in R. O. Smitherman, W.L. Shelton, J.H. Grovers Eds. Culture of exotic fishes. Symposuim Proceedings. Fish Culture Section, American Fisheries Society, Auburn, Alabama. 27. Swanson, H.E., E. Vander Werfften and J.J. Bosch, The early androgen syndrome : Effects of prenatal testosterone propionate. Acta, Endocrinol., 50: Tayamen, M.M. and W.L. Shelton, Inducement of sex reversal in Sarotherodon niloticus (Linnaeus). Aqua., 14: William, L.G., S.F. Martin, M.C. Wilfrido and B.S. Carl, Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in androgens. Aqua., 178(3-4): Wolf, C.J., A.L. Gerald, L. Earl and J.R. Gray, Interactive effects of vinclozolin & testosterone propionate on sexual differentiation of the rats. Toxicological Sciences, 78: Yamamoto, T., Sex differentiation in tilapia. in Fish Physiology, pp: Ed. By W.S. Hoar and D.Y. Randall.,.3, Academic press, New York. 32. Yamamoto, T. and T. Kajishima, Sex hormonic induction of reversal of sex differentiation in gold fish and evidence of the male heterogamety. J. Exp. Zool., 162: Yamazaki, K., Application of hormones in fish culture.j.fis.board Can., 33: Yeung, W.S.and S.T. Chan, A radioimmunoassay study of the plasma levels of sex steroids in the protandrous, sex -reversing fish Rhabdosargus sarba (Sparidae). Gen. Comp. Endocrinol., 66(3): Yoshikawa, H. and M. Oguri, 1978a. Sex differentiation in a cichlid,tilapia zillii. Bull. Jap. Soc. Fish.., 44: Yoshikawa, H. and M. Oguri, 1978b. Effects of steroid hormones on the sex differentiation in a cichlid, Tilapia zillii. Bull. Jap. Soc. Sci., Fish., 4(10):