Aquacult Int (2007) 15:109–119 DOI 10.1007/s10499-006-9071-0 ORIGINAL PAPER Masculinization of Convict Cichlid (Cichlasoma nigrofasciatum) by immersion in Tribulus terrestris extract Şehriban Çek Æ Funda Turan Æ Esin Atik Received: 5 June 2006 / Accepted: 22 December 2006 / Published online: 6 March 2007
Springer Science+Business Media B.V. 2007 Abstract We have examined the effects of Tribulus terrestris (TT), a non-toxic herb, on sex reversal in the Convict Cichlid Cichlasoma nigrofasciatum with the aim of introducing a new environmentally friendly method for masculinization in C. nigrofasciatum. TT is a natural plant product that elevates the testosterone levels in humans and animals. Different concentrations (0.0, 0.10, 0.20 and 0.30 g/l) of TT extract were tested for their effect on sex reversal in C. nigrofasciatum by immersing newly hatched offspring once weekly for 2 months in TT extract. Of the dosages used in the present study, 0.30 g/l TT was the most effective in terms of masculinization, resulting in a maximum male ratio of 87.23% (P < 0.001). Sex ratios of 79 and 85% at 0.10 and 0.20 g/l TT, respectively, were also significantly different from the expected 1:1 ratio (P < 0.001). Histological examinations revealed that the testes of fish treated with TT extract contained all stages of spermatogenesis, clearly demonstrating that the administration of TT extract to C. nigrofasciatum stimulated spermatogenesis. Total survival rates in all treatments and the control were uniformly high, ranging from 88.57 to 90% (P > 0.05). We concluded that TT had no negative effect on the survival rate of C. nigrofasciatum. In addition, all groups of TT-treated fish exhibited successful growth acceleration compared to the control group, but only the 0.30 g/l TT treatment significantly improved the growth rate of C. nigrofasciatum. (P < 0.01). Sex reversal in C. nigrofasciatum demonstrated that TT-treated 0-day-old larvae showed successful sex reversal, spermatogenesis and a better growth rate than untreated progenies. Keywords Cichlasoma nigrofasciatum Æ Growth Æ Histology Æ Sex reversal Æ Tribulus terrestris Ş. Çek (&) Æ F. Turan Faculty of Fisheries and Aquaculture, Mustafa Kemal University, 31040 Seinyol Antakya-Hatay, Turkey e-mail: scek@mku.edu.tr E. Atik Faculty of Medicine, Department of Pathology, Mustafa Kemal University, Antakya-Hatay, Turkey 123 110 Şehriban et al. Introduction Gokshura, Tribulus terrestris L. (Zygophyllaceae), is an herb that is widely distributed in China, Japan, Korea, the western part of Asia, the southern part of Europe and Africa. It has been shown to raise testosterone levels safely and naturally and is rumoured to be the secret behind the success of many top Bulgarian weightlifters (Bucci 2000). The administration of T. terrestris L. (TT) to humans and animals improves the libido and spermatogenesis (Tomova et al. 1981). In humans, it has been used to treat impotence and has been found to increase testosterone levels and improve athletic performance (Adimoelja and Adaikan 1997; Adimoelja 2000; Gauthaman et al. 2000; Bucci 2000; Adaikan et al. 2000 and Gauthaman et al. 2002). TT contains a number of different substances known as steroidal saponins. Protodioscin, the most dominant saponin in TT, is thought to be main substance responsible for increasing testosterone production (Ganzera et al. 2001). Protodioscin has also been found to increase the levels of dehydroepiandrosterone (Adimoelja and Adaikan 1997), dihydrotestosterone and dehydroepiandrosterone sulphate (Gauthaman et al. 2000). Cichlids are an important group of relatively large and often colourful aquarium fishes. The family cichlidae comprises about 105 genera and 1300 species, making it the second largest perciform family (Nelson 1994). Convict Cichlid Cichlasoma nigrofasciatum is a medium-sized cichlid from Central America (Axelrod et al. 1971). In this study it was used as a model fish because of its wide range of tolerance to water hardness, pH and temperature. It is also easily obtainable and can be propagated inexpensively (Winemiller and Taylor 1982; Colgan and Salmon 1986; Wisenden 1994; Fraser 1996). Under optimum conditions the fish produce large numbers of viable eggs (100–800 per batch). The development period from fertilization to hatching is very brief, approximating 3 days at 27C (Piron 1978; present study). This species also shows notable reproductive features, such as a complex social and breeding behaviour, which includes parental care and, most importantly, a high spawning rate (about every 20 days during a9-month period) (Colgan and Salmon 1986; Fraser 1996; Meijide and Guerrero 2000). The sex ratio of cichlid fish has great significance in aquaculture because uncontrolled reproduction of this group in production ponds is one of the most serious limitations in cichlid culture. Males grow faster than females (Myers et al. 1995; Tarık Ezaz et al. 2004); therefore, the maintenance and breeding of male populations have generated a great amount of interest in terms of commercial applications. Cichlid fish can be masculinized by direct synthetic hormonal treatment that is efficient and straightforward (Pandian and Sheela 1995; George and Pandian 1996; Gale et al. 1999). However, synthetic hormones are more expensive than plant extracts, and their administration in fish is time-consuming and labour-intensive and requires specialist expertise. Furthermore, synthetic hormones have been reported to have the potential to accumulate in the sediment water and aquatic biota (Contreras-Sanchez et al. 2001; Çek et al. 2004). At the present time, there are no data available on the accumulation of protodioscin in the water sediment or on the toxicity of TT in fish. In humans, research on the application of a dry powder extract of Tribulus terrestris has been carried out in the context of treating clinical symptoms of ageing men. It has been reported that protodiocin, the active component of TT extract, can be converted to 123 The effect of Tribulus terrestris on Convict Cichlid 111 dehydroepiandrosterone (DHEA). Tribulus terrestris extract, which contains a non-hormonal Phyto-DHEA, is the complementary drug of choice to cure sexual dysfunction in men (Adimoelja 2000; Adimoelja et al. 2005). An alternate technique for commercially producing all-male fish populations would be to use plant extracts. Therefore, the objective of the present study was to investigate the effect of TT on sex reversal and growth rate in C. nigrofasciatum. Material and methods Plant material, preparation of herbal extracts and experimental design The TT extract (origin: Bulgaria) was purchased from Dietharmonie Medicinal Plants (France). The aqueous extracts of TT were prepared by boiling 36 g of the pure and finely ground extract of TT in 180 l distilled water for 30 min and then filtering it through a Whatman paper filter twice (Gauthaman and Adaikan 2005). This solution was not a stock solution but was prepared on eight separate occasions (weekly for 60 days; each aquaria contained 20 l); that is to say, there were three replicates for the TT treatment, 36 g of TT were used per immersion and the larvae were exposed eight times (Gauthaman and Adaikan 2005). The fish remained in the solution for an entire week, at which time the water of the aquaria was changed entirely (every 7 days for 60 days). TT is soluble in water and, therefore, the immersion treatment was used as an alternative to the more common, oral/diet application method. The immersion method also ensures synergic induction, is cheaper than dietary treatment and requires almost no skill (Pandian and Sheela 1995). In the diet supplementation technique, the hormone is degraded in the digestive tract. In addition, its purity varies as well as its solubility, which is dependent on the solvent used; as such, the uniformity of its distribution in the feed may vary. Size hierarchy may lead to differential feed uptake and hence differential hormone intake (Pandian and Sheela 1995). A stock of C. nigrofasciatum, comprising males (mean weight: 3.15 ± 0.073 g; mean length: 6.35 ± 0.15 cm) and females (mean weight: 2.45 ± 3 g; mean length: 5.50 ± 0.95 cm) was procured from a local ornamental fish dealer. Variability in sex ratios among individual spawns in cichlids has been observed by Shelton et al. (1983). Therefore, one male and female (all of the larvae used in these treatment studies were obtained from one pair of spawning adult fish) from this stock were placed in an aquarium containing recirculating water (26 ± 1C) and exposed to a 12 (light):12 (dark) photoperiod. Once a female had spawned—in a mussel shell—the eggs hatched within approximately 3 days, and larvae were immediately removed from the aquarium, counted, measured and placed in 12 small glass aquaria, each containing 20 l of water that was continuously aerated with a 4-cm air stone. A total of 420 fry were separated into four equal treatment groups (35 fry/aquarium, three aquariums/treatment). The larvae were fed three times a day with commercial flake food supplemented with freshly hatched artemia salina and tubifex throughout the experimental period of 3.5 months. The effects of different concentrations [0.0 g/l (control); 0.10, 0.20 and 0.30 g/l (experimental); once weekly for 60 days] of TT on the sex ratio and growth rate were investigated in C. nigrofasciatum in four trials. 123 112 Şehriban et al. Sampling and histological procedures At the end of the experiment, 3.5-month-old fish were anaesthetized in 2-phenoxethanol (0.04%) and counted with the aim of assessing the survival rate, and whole body weights and lengths were recorded. The head and tail of the fry were cut off, and the body fixed in 10% neutral buffered formalin, dehydrated, embedded in paraffin, sectioned (5-lm thicknesses) and stained with haematoxylin and eosin for histological examination (Çek et al. 2001; Çek 2006). The stage of testes and ovary development was determined for each fish. Classification was based on the histological criteria adapted from Nicholls and Graham (1972), Grier (1981, 1993) and Bromaga and Cumaranatunga (1987). Statistical analysis Differences between groups in terms of sex ratio of the offspring were determined on the basis of gonad histology and secondary sex characteristics and analyses by the chi-square (v2) test (Zar 1984). The secondary sex characteristics used included: males grow larger but are less colourful than females and they develop longer dorsal and anal fins as well as a fatty lump on their foreheads; females develop a pinkish colouration at the base of the dorsal and caudal fins and may also develop a fatty lump on their foreheads but one that is smaller than that found in males. Differences in mortality and differences in body weight and length between groups were tested with the Kruskal-Wallis one-way analysis of variance by ranks (SPSS ver. 10.0 for Windows; SPSS, Chicago, Ill.) followed by the Duncan non-parametric multiple comparison procedure. Results Effect of TT on the sex ratio of C. nigrofasciatum In the present investigation, we achieved 87.23% masculinization in C. nigrofasciatum by immersing 0-day-old fry for 60 days in water containing 0.30 g TT per litre. All of the fish groups treated with various doses of TT showed marked masculinizing effects during the 60-day experimental period. At the termination of the experiments, this effect had resulted in a statistically significant difference in the sex ratio (P < 0.001) compared to the sex ratio of fry in the first series of the experiments (0 g/l TT), which served as control groups and which had nearly the expected sex ratio of 1:1 (male:female) (Table 1). The sex ratio observed in 93 fish in the second series of experiment (0.10 g/l TT treatment groups) was 74:19 (male:female), while in the third series of experiments (0.20 g/l TT treatment groups), it was 80:14 (male:female). The maximum ratio of male fish was obtained in the 95 fish in the last series of experiments, which had been given the highest dose of TT (0.30 g/l TT): the sex ratio observed in this group was 82:11:2 (male:female:intersex). These results were interpreted as an indication that increasing concentrations of TT caused an increase in the number of males produced. 123 The effect of Tribulus terrestris on Convict Cichlid 113 Table 1 Effects of treatment with Tribulus terrestris (TT) on sex ratio in Cichlasoma nigrofasciatum Dosage TT (g/l) Treatment duration (day) Sex distributions (male:female:intersex; M:F:I) Sex ratio (%) (M:F) v2 0 0.1 0.2 0.3 60 60 60 60 42:51 (n = 93) 74:19 (n = 93) 80:14 (n = 94) 82:11:2 (n = 95) 45.16:54.84 79.57:20.43 85.11:14.89 87.23:12.77 _ 32.527*** 46.340*** 54.204*** *** Sex ratio significantly different from expected 1M:1F (P < 0.001, v2 values are for comparisons of sex ratios with the control *,P<0.05; **,P<0.01; ***, P<0.001), TT, Tribulus terrestris. In all groups, treatment began on the first day of hatching. Effect of TT on gonad development of C. nigrofasciatum Histological examination of the TT-treated testes and ovaries revealed no damage to the testicular and ovarian structure. All spermatogenesis stages were present in both the control and TT-treated male testes. However, spermatogenesis was more advanced among the TT-treated groups of C. nigrofasciatum compared to the control groups (unpublished data). It would appear that the histological response of the testis in all of the TT treatment groups included an increased number of spermatogenetic cysts and an abundance of the late stages of spermatogenesis. These testes contained a preponderance of spermatozoa in the lobular lumen. All stages of spermatogenesis, including ruptured spermatozoa, were detected in the sperm ducts (Figs. 1, 2). Lobules containing numerous spermatocytes from early stages (spermatogonia) to complete spermatogenesis (spermatocytes, spermatids and spermatozoa) were observed (Fig. 1). In comparison, in the control groups, free spermatozoa were only occasionally recorded, and the testis contained mostly spermatogonia and spermatocytes (Fig. 3A, B). In the last series of experiments (the 0.30 g/l TT treatment), two inter-sex fish were recorded (Fig. 2) in which the TT-treated male possessed two testes’ lobes that consisted of many lobules with numerous sperm and a few primary ova situated on the sperm duct of only one testicular lobe. These morphological characteristics provided direct evidence that this TT-treated male was likely a sex reversal with a female genotype (Fig. 2). Despite differences in the sex ratio, spermatogenesis and growth rate, no marked differences in the structure of the ovaries structure were found between the control and TT-treated groups. Survival and growth of TT-treated fish Table 2 shows the survival and growth rates of both control and TT-treated C. nigrofasciatum in terms of total body length and body weight. Total survival rates in all treatment and control experiments were uniformly high, ranging from 88.57 to 90.47% (P > 0.05). All groups of TT-treated fish exhibited a successful growth acceleration compared to the control group, but only the 0.3 g/l TT treatment significantly increased the growth rate of C. nigrofasciatum (P < 0.001; Fig. 4). In the last series of the experiment, total body length and weight (4.65 ± 0.38 cm and 2.35 ± 0.24 g, respectively) were significantly increased compared to the controls (3.72 ± 0.1 cm; 1.41 ± 0.14 g). This indicates that TT has no 123 114 Fig. 1 A portion of the TT-treated male Convict Cichlid’s (Cichlasoma nigrofasciatum) gonad showing all stages of spermatogenesis and cell divisions within the lobules. L Lumen, SG spermatogonia, PS primary spermatocytes, SS secondary spermatocytes, ST spermatids, S spermatozoa. Magnification: 1000·; stains: haematoxylin and eosin Şehriban et al. SS SG S PS L ST Fig. 2 Haematoxylin-eosinstained cross section of the testis of a male Convict Child treated for 60 days in 0.3 g/l TT. Note the presence of oocytes at the perinucleolar stage (stage 2 oocytes; STO), showing direct evidence that this TT-treated male may be a sex reversal with the female genotype. PS Primary spermatocytes, SS secondary spermatocytes, S spermatozoa. Magnification: 600· S SS PS STO negative effect on the survival rate of C. nigrofasciatum but that it has the ability to increase total body weight and length at the tested concentrations. Discussion and conclusion The goal of the present study was to find an effective masculinization method for sex reversal and growth performance in fish culture that is cheap, environmentally friendly, easy to use and more effective than those methods currently in use. To this end, we used the Convict Cichlid as a model fish. We found that TT extract alone was effective at various dose levels in increasing the proportion of males in the population, advancing spermatogenesis and improving growth performance in C. nigrofasciatum. Prior to this study, our group had obtained similar results using TT extracts to produce a male population in Poeciliata reticulata (Çek et al. 2007). To our knowledge, this study documents the first reported investigation aimed at evaluating TT as a potent masculinizing agent in C. nigrofasciatum. Survival ratios at the termination of the experiment in the controls were similar to those observed in the TT-treated groups: there were no significant dose-related inter-group differences noted. 123 The effect of Tribulus terrestris on Convict Cichlid Fig. 3 Cross section of male gonads from control groups. (A) Although all stages of spermatogenesis were detected, ruptured spermatozoa were only occasionally recorded. Magnification: 400·; stains: haematoxylin and eosin. (B) The testis contained mostly spermatogonia, spermatocytes and spermatids. Magnification: 1000·; stains: haematoxylin and eosin. SG Spermatogonia, PS primary spermatocytes, SS secondary spermatocytes, ST spermatids, S spermatozoa 115 A SG ST PS SS B ST PS SS SG Table 2 Effects of treatment with TT on the survival and growth rate in C. nigrofasciatum Dosage of TT (g/l) Survival rate (%) Total length (cm) Body weight (g) 0 0.1 0.2 0.3 88.57 88.57 89.52 90.47 3.72 3.77 3.85 4.65 1.41 1.43 1.51 2.35 ± ± ± ± 1.65 3.29 2.52 3.43 a a a a ± ± ± ± 0.10 0.22 0.18 0.38 a a a b ± ± ± ± 0.14 0.29 0.18 0.24 a a a b Values (mean ± SE of triplicate trials) followed by different letters within the same column are significantly different (P < 0.01) The work of Adimoelja (2000) in Indonesia and Adaikan et al. (2000) in Singapore have presented evidence that TT is not toxic to humans and rabbits, respectively. Tapia et al. (1994), Waller and Yamasaki (1996) and Aslani et al. (2004) studied the toxicity of TT in livestock and concluded that the consumption of TT by livestock led to the photosensitization syndrome known as yellow thick head. In these studies, TT was not purified, and it was fed to the animals ad libitum for at least 2 months. Photosensitization has not been observed in humans and is highly unlikely in fish at the recommended dosage (personal communication from A. Adimolja). Kavitha and Jagadessan (2002, 2003) studied the role of the TT extract on mercury-intoxicated mice, Mus musculus. In their study, a lethal dose of mercuric chloride was administrated through the drinking water to female mice every day for 45 days. Its toxicity altered the histoarchitecture of the large intestine. During the 123 Total Weight (g) 5 6 4,5 5 Body Length (cm) 5 4 3 4 3,5 4 3 2,5 3 2 2 2 1 1 0 1,5 1 0,5 0 0 0,1 0,2 0,3 Tribulus terrestris (g/l) Total Length (cm) Total Weight (g) 0 0 0,1 0,2 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 Body weight (g) Total Length (cm) 6 Body Length (cm) Fig. 4 Effects of TT on total body length and weight of C. nigrofasciatum (n = 100; P < 0.001) Şehriban et al. Body Weight (g) 116 0,3 Tribulus terrestris (g/l) recovery period, the mice were dosed with a TT extract of different solvent fractions for 15 days; these mice showed a complete regeneration of the large intestine from the toxic effect of the mercury (Kavitha and Jagadessan 2002, 2003). In the present study photosensitization was not observed, and the survival ratios of the TT-treated groups of fish were not different than that of the control groups. However, the toxicity of TT in fish (if present) needs to be studied. We also determined in this study that TT was not harmful by inhalation and that absorption through the skin did not cause irritation. Synthetic androgens are harmful when inhaled, ingested and/or absorbed through the skin and can cause irritation. Although the present research provides evidence that TT treatments result in a high rate of masculinization, whether this potency is caused by increases in androgens or testosterone cannot be deduced from the present results, as we did not measure plasma testosterone level during the experiment. However, the present results are consistent with reported results from studies with such fish species as Onchorynchus tshawtscha, Cyprinus carpio and Cichlasoma nigrofasciatum, all of which were treated with synthetic hormone (Baker et al. 1988; Ali and Rao 1989; George and Pandian 1996). However, to the best of our knowledge, prior to the present research, the potency of TT/or a plant extract as masculinizing agent in fish has not been reported in fish. We found that testes development in the control group was normal: males in the control group had relatively smaller gonads that mostly contained spermatogonia and spermatocytes. The sex ratio of male to female in this group was 45.16:54.84. In contrast, the groups treated with various doses of TT showed marked masculinizing effects, and their testes showed advanced development, both morphologically and histologically. Spermatozoa development could be classified into five stages in all TT-treated groups based on histological examination, a finding consistent with those of Grier (1981), Fishers (2003) and Meijide et al. (2005). Similar to results observed in humans (Seth 1974; Produce et al. 1983), immature sheep (lambs) (Georgiev et al. 1988), rats (Dimitrov et al. 1987) and fish (Çek et al., 2007), the testes in the treated fish showed a significantly improved spermatogenesis compared with the controls. Among the fish subjected to the higher dosage, two of the treated individuals were found to develop intersex gonads. These could have appeared either in response to a sub-optimum intensity of the treatment (e.g. anabantids) or as a simultaneous occurrence among the fish treated at super-optimal doses. Reports on simultaneous occurrences of intersex fish are available for cichlids (Pandian and Sheela 1995). Moreover, paradoxical feminizing effects of high dosages of androgens have been found in Tilapia mossambicus (Nakamura 1975), Pikeperch, Stizostedion lucioperca (Demska-Zakes and Zakes 123 The effect of Tribulus terrestris on Convict Cichlid 117 1997), hybrid Red tilapia Oreochromis niloticus x Oreochromis mossambicus (Manosroi et al. 2004). Pandian and Sheela (1995) have reviewed this phenomenon. A previous attempt by Gautman et al. (2002) to determine the aphrodisiac properties of TT in normal and castrated rats yielded successful results. They concluded that the TT extract increases testosterone levels in rats. In the present study, the growth rate of fish treated with TT was found to be faster than that of the controls. The effects of the TT extract on body weight have been studied by Georgiev et al. (1988) in immature sheep and by Gauthaman et al. (2002) in rats. Both authors found an increase in body weight and sexual activity and spermatogenesis. While these findings are not contradictory with the present results, there is a lack of information in the literature on the effects of a plant extract on sex-reversal, gonad development and growth performance in a fish species. We demonstrate here, for the first time, that TT extracts are potent and able to induce a high rate of sex-reversal – 87.23%–and lead to a significant accelerated growth rate in C. nigrofasciatum. TT treatment is a more effective method than the administration of synthetic hormone in terms of effecting sex reversal and is more environmental friendly and cheaper (only 1 ml 17a-methyl testosterone is £78.03; 100 capsules of TT is only $8.99). In addition, the persistence and fate of synthetic hormones and hormone metabolites in fish, water and sediment may represent potential environmental and health risks that have to be considered when using hormonal sex control technology (Contreras-Sanchez et al. 2001). Tribulus terrestris can be applied with ease to a large number of individuals simultaneously. The use of TT as an alternative method to produce preponderantly male populations of C. nigrofasciatum may address environmental safety issues. Fish offered to the consumer will not be treated with synthetic hormones, and producers may have an alternative method for producing monosex populations based on natural products. The findings from this study add further support to the effect of TT on growth and its testosterone-releasing property. Future studies aimed at measuring the amount of testosterone levels after TT treatment in C. nigrofasciatum may provide more conclusive evidence in terms of the effects of TT on the sex ratio and whether it can be successfully used as an agent and/or a method in fish culture. Further investigations are necessary to determine the effects of TT on other cultivable fish species. Acknowledgements The Mustafa Kemal University supported this study financially. We would like to wholeheartedly thank Dietharmonie, France for providing the TT extract that was used in this study. We also thank Dr. Haydar Fersoy and Dr. Yavuz Mazlum for reading the first draft of this manuscript. References Adaikan PG, Gauthaman K, Ng RNV, Prasad SC (2000) Proerectile pharmacological effects of Tribulus terrestris on the rabbit corpus cavernosum. Ann Acad Med Singapore 29(1):22–26 Adimoelja A (2000) Photochemical and the breakthrough of traditional herbs in the management of sexual dysfunctions. Int J Androl 23(2):82–84 Adimoelja A, Adaikan PG (1997) Protodioscin from herbal plant Tribulus terrestris L. improves male sexual functions possibly via DHEA. Int J Impot Res 9:64 Adimoelja A, Sartono S, Soedjono J (2005) Phyto-DHEA treatment, an alternative option for aging men. Int J Androl 28(Suppl 1):25 Ali PK, Rao GPS (1989) Growth improvement in carp, Cyprinus carpio (Linnaeus), sterilized with 17a-methyl testosterone. Aquaculture 76:157–167 Aslani MR, Movassaghi AR, Mohri M, Ebrahim-pour V, Mohebi AN (2004) Experimental Tribulus terrestris poisoning in goats. Small Ruminant Res 51:261–267 123 118 Şehriban et al. Axelrod HR, Emmens CV, Sculthorpe D, Wingler WV, Pronek N (1971) Exotic tropical fishes. TFH Publ, Jersey City Baker IJ, Solar II, Donaldson EM (1988) Masculinization of Chinook salmon (Onchorynchus tshawtscha) by immersion treatments using 17a-methyl testosterone around the time of hatching. Aquaculture 72:359–367 Bromaga NR, Cumaranatunga PRT (1987) Oocyte development in the rainbow trout with special reference to vitellogenesis and atresia. In: Idler DR, Crim LW, Walsh JM (eds) Proc 3rd Int Symp Reprod Physiol Fish. St John’s, Newfoundland, p 194 Bucci LR (2000) Selected herbals and human exercise performance. Am J Clin Nutr 72:624–636 Colgan PW, Salmon AB (1986) Breeding experience and parental behaviour in convict cichlids Cichlasoma nigrofasciatum, Behav Process 13(1–2):101–118 Contreras-Sanchez WM, Fitzpatrick MS, Schreck CB (2001) Fate of methyl testosterone in the pond environment: detection of Mt in pond soil from a CRSPsite. In: Gupta A, McElwee K, Burke D, Burright J, Cummings X, Egna H (eds) 18th Annu Tech Rep. PD/A CRSP (Oregon State University, Pond Dynamics/Aquaculture Collaborative Research Support Program), Corvallis, Oregon, pp 79–82 Çek Ş (2006) Early gonadal development and sex differentiation in rosy barb (Puntius conchonius). Anim Biol 56:335–350 Çek Ş, Bromage NR, Randall C, Rana K (2001) Oogenesis, hepatosomatic indexes, and sex ratio in Rosy barb (Puntius conchonius). Turk J Fish Aquat Sci 1:133–141 Çek Ş, Yıldırım Y, Şereflis¸ an H, Akyurt I_ (2004) Sex control in aquaculture. In: Harmancıoğlu O, Fıstıkoğlu Y, Dalgılıç D, Gul A (eds) Water resources management; risks and challenges for the _ 21st century. Izmir, Turkey, pp 343–353 Çek Ş, Turan F, Atik E (2007) The effects of gokshura, Tribulus terrestris on sex differentiation of guppy, Poecilia reticulata. Pakistan J Biol Sci 10(5): 718–725 Demska-Zakes K, Zakes Z (1997) Effect of 17 a-methyl testosterone on gonadal differentiation in pikeperch, Stizostedion lucioperca L. Aquac Res 28:59–63 Dimitrov M, Georgiev P, Vitanov S (1987) Use of Tribestan on rams with sexual disorders. Vet Med Nauki 24(5):102–110 Fishers L (2003) Comparison of testes structure, spermatogenesis and spermatocytogenesis in young, aging and hybrid cichlid fish (Cichlidae, Teleostei). J Morphol 256(3):285–300 Fraser SA (1996) The influence of predators on adaptation behaviour in adult convict cichlid Cichlasoma nigrofasciatum. Can J Zool 74:1165–1173 Gale WL, Fitzpatrick MS, Lucero M, Contreras-Sanchez WM, Schreck CB (1999) Masculinization of Nile tilapia (Oreochromis niloticus) by immersion in Androgens. Aquaculture 178:349–357 Ganzera M, Bedir E, Khan IA (2001) Determination of steroidal saponins in Tribulus terrestris by reversed-phase high-performance liquid chromatography and evaporative light scattering detection. J Pharm Sci 90:1752–1758 Gauthaman K, Adaikan PG (2005) Effects of Tribulus terrestris on nicotinamide adenine dinucleotide phosphate diaphorase activity and androgen receptors in rat brain. J Ethnopharmacol 96(1–2):127–132 Gauthaman K, Adaikan PG, Prasad RNV, Goh VHH, Ng SC (2000) Changes in hormonal parameters secondary to intravenous administration of Tribulus terrestris extract in primates. Int J Impot Res 12:6 Gauthaman K, Adaikan PG, Prasad RNV (2002) Aphrodisiac properties of Tribulus terrestris extract (Protodioscin) in normal and castrated rats. Life Sci 71(12):1385–1396 George T, Pandian TJ (1996) Hormonal induction of sex reversal and progeny testing in the zebra cichlid Cichlasoma nigrofasciatum. J Exp Zool 275:374–382 Georgiev P, Dimitrov M, Vitanov S (1988) The effect of the preparation ‘Tribestan on the plasma concentration of testosterone and spermatogenesis of lambs and rams. Vet Sb 3:20–22 Grier HJ (1981) Cellular organization of the testis and spermatogenesis in fishes. Am Zool 21:345–357 Grier H (1993) Comparative organization of sertoli cells including the sertoli cell barrier. In: Rusell LD, Griswold MD (eds) The sertoli cell book. Cache River Press, St. Louis, Mo., pp 703–739 Kavitha AV, Jagadessan G (2002) In vivo studies on the role of Tribulus terrestris extract on mercury intoxicated mice, Mus musculus (Linn.) large intestine – a histological survey. J Exp Zoo India 6(2):213–219 Kavitha AV, Jagadessan G (2003) Histopathological alterations in small intestine of mercury intoxicated mice, Mus musculus (Linn.) in response to Tribulus terrestris (Zygophyllaceae) extract. Pollen Res 23(3):343–347 123 The effect of Tribulus terrestris on Convict Cichlid 119 Manosroi J, Petchjul K, Manosroi A (2004) Effect of fluoxymesterone fish feed granule on sex reversal of the hybrid, Thai red tilapia (Oreochromis niloticus Linn. · Oreochromis mossambicus Linn). Asian Fish Sci 17:323–331 Meijide FJ, Guerrero GA (2000) Embryonic and larval development of a substrate-brooding cichlid Cichlasoma dimerus (Heckel, 1840) under laboratory conditions. J Zool 252:481–493 Meijide FJ, Lo Nostro FL, Guerrero GA (2005) Gonadal development and sex differentiation in the cichlid fish Cichlasoma dimerus (Teleostei, perciformes): a light and electron-microscopy study. J Morphol 264(2):191–210 Nelson J (1994) Fishes of the world, 3rd edn. Wiley, New York Myers JM, Penman J, Rana KJ, Bromage NR, Powell SF, McAndrew BJ (1995) Applications of induced androgenesis with tilapia. Aquaculture 137(1–4):150 Nakamura Y (1975) Dosage-dependent changes in the effect of oral administration of methyl testosterone on gonadal differentiation in Tilapia mossambicus. Bull Fac Fish Hokkaido Univ 26:99–108 Nicholls TJ, Graham GP (1972) The ultra structure of lobule boundary cells and leyding cell homologs in the testis of a cichlid, Cichlasoma nigrofasciatum. Gen Comp Endocrinol 19:133–146 Pandian TJ, Sheela SG (1995) Hormonal induction of sex-reversal in fish. Aquaculture 138:1–22 Piron RD (1978) Breeding the convict cichlid (Cichlasoma nigrofasciatum) for use in laboratory fish toxicity tests. J Fish Biol 13:119–122 Produce M, Tsvetkov D, Nalbanski B (1983) Clinical trial of the preparation Tribestan in infertile men. Akush Ginekol 22(4):326–329 Seth SD (1974) Preliminary pharmacological investigations of Tribulus terrestris. Linn (Gokhru). Indian J Med Sci 28(9):377–380 Shelton WL, Meriwether FH, Semmens KJ, Calhoun E (1983) Progeny sex ratio from intraspecific pair spawning of Tilapia aureus and Tilapia nilotica. In: Fishers L, Yaron Z (eds) International Symposium on Tilapia in Aquaculture. Tel Aviv University, Tel Aviv, pp 270–280 Tapia MO, Giordano MA, Gueper HG (1994) An outbreak of hepatogenous photosensitization in sheep grazing Tribulus terrestris in Argentina. Vet Hum Toxicol 36(4):311–313 Tariq Ezaz M, Mayers JM, Powell SF, McAndrew BJ, Penman DJ (2004) Sex ratio in the progeny of androgenic and gynogenetic YY male Nile tilapia, Oreochromis niloticus L. Aquaculture 232(1–4):205–214 Tomova M, Gjulemetova R, Zarkova S, Peeva S, Pangaroya T, Simova M (1981) Steroidal saponins from Tribulus terrestris L. with a stimulating action on the sexual functions. In: 1st Proc Int Conf Chem Biotechnol Biol Active Nat Products. Varna, pp 298–302 Waller GR, Yamasaki K (1996) Saponins used in food and agriculture. Advances in experimental medicine and biology. Plenum Press, New York, pp 381–382 Winemiller KO, Taylor DH (1982) Inbreeding depression in the convict cichlid, Cichlasoma nigrofasciatum (Baird and Girard). J Fish Biol 21:399–402 Wisenden BD (1994) Factors affecting reproductive success in free ranging convict cichlids. Can J Zool 72:2177–2185 Zar H (1984) Biostatistical analysis. Prentice-Hall, Upper Saddle River 123