Preliminary Investigation of Tribulus terrestris (Linn., 1753) Extracts as Natural Sex Reversal Agent in Oreochromis niloticus (Linn., 1758) Larvae  

B.O. Omitoyin , E.K. Ajani , H.O. Sadiq
Department of Aquaculture and Fisheries Management, University of Ibadan, Nigeria
Author    Correspondence author
International Journal of Aquaculture, 2013, Vol. 3, No. 23   doi: 10.5376/ija.2013.03.0023
Received: 08 Jul., 2013    Accepted: 22 Jul., 2013    Published: 23 Jul., 2013
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Omitoyin, 2013, Preliminary Investigation of Tribulus terrestris (Linn., 1753) Extracts as Natural Sex Reversal Agent in Oreochromis niloticus (Linn., 1758) Larvae, International Journal of Aquaculture, Vol.3, No.23 133-137 (doi: 10.5376/ija.2013. 03.0023)


Synthetic sex reversal hormone used in controlling Tilapia population under culture condition has a perceived public health risk. This study therefore examined the prospect of utilization Tribulus terrestris as sex reversal agent in Oreochromis niloticus. Five pairs of Oreochromis niloticus (350±25.8) g in a ratio of 1:1 male to female were used for fry production. Four hundred and fifty day-old fry were randomly distributed into 18 experimental tanks (30 L capacity) and fed 50% crude protein diets containing 0.0 g, 1.0 g, 1.5 g, 2.0 g, and 2.5 g per 1 kg of basal feed of T. terrestris extract. Diet six contained 50 mg/kg of basal feed of 17-α-methyltestosterone served as control. Data collected includes, water quality (Temperature, pH and Dissolved oxygen), Growth performance (Mean Weight Gain (MWG), Food Conversion Ratio (FCR) and Specific Growth Rate (SGR)), survival rates at the fry and fingerlings stage and sex ratio. Data collected were subjected to 2-way analysis of variance. Temperature and pH ranged between 26 ℃ to 28 ℃ and 7.7 to 8.2 respectively. Dissolved oxygen varied from 5.13 mg/L to 9.12 mg/L. The mean weight gain of fry ranged from (0.73±0.08) g to (0.92±0.1) g and fingerlings from (14.35±1.26) g to (18.29±1.59) g which was significantly different across the treatments (p<0.05). FCR recorded for fry ranged from 1.53±0.17 to 2.12±0.14 and SGR, from 14.477±0.15 to 15.257±0.25. There was no significant difference in FCR and SGR across the treatments for fingerlings. Survival rates ranged from 80% to 89.3%. Percentage of males in treated fish increased as concentration of T. terrestris increased with T5-2.5g (85.7±1.3) %.

Sex reversal; Population control; Tribulus terrestris; Oreochromis niloticus

Tilapias constitute one of the most productive and internationally traded food fish in the world (Modadugu and Belen 2004; Lim and Webster, 2006) and one of the major sources of protein to most developing countries. Characteristics that make Tilapia the best choice for farmers include; resistance to disease, tolerance to a wide range of environmental conditions, ability to convert efficiently organic, domestic and agricultural wastes, into high quality protein, good growth rates and ease of growth in intensive culture (Penna-Mendoza et al., 2005; Altun et al., 2006). Despite having many good characteristics, one of the major drawbacks in commercial tilapia production is its precocious maturity and the following uncontrolled reproduction, resulting in increasing competition for feed followed by stunted growth and low commercial value (Jegede and Fagbenro, 2007; Wassermann and Afonso, 2003). Fast growth rate at high density, acceptability to artificial feed and controlled reproduction are some of the most important characteristics in a successful fish culture.

In populations of tilapia, males grow faster and are more uniform in size than females (Tariq-Ezaz et al., 2004). For this reason, the farming of monosex populations of tilapias, which is achieved either by manual sexing, direct hormonal sex reversal, hybridization or genetic manipulation, has been reported as a solution to the problem of early sexual maturation and unwanted reproduction (Gupta and Acosta, 2004). The production of monosex populations has been successfully achieved by oral administration of natural or synthetic steroidal hormone to masculinize or feminize sexually undifferentiated fry (Pandian and Sheela, 1995). This technique has been widely used. However, increasing consumer rejection to the use of hormone in food production has limited this application.
Tilapia can be masculinized by direct synthetic hormonal treatment that is efficient and straightforward (Gale et al., 1995). However, synthetic hormones are more expensive than local plant extracts; also their administration in fish is time-consuming and labour-intensive and requires expertise. Further, synthetic hormones accumulate in the sediment water and aquatic biota (Cek et al., 2004) and there are concerns about consumer perceptions of eating hormone treated fish (Phelps and Carpenter, 2002).
Tribulus terrestris is known to elevate the testosterone levels in humans and animals. In humans, it has been used to treat impotence and has been found to increase testosterone levels (Adaikan et al., 2000; Gauthaman et al., 2002). T. terrestris contains protodioscin, a saponin, which is considered the main substance responsible for increasing testosterone production (Ganzera et al., 2001). There is no report on the accumulation of protodioscin in the sediment water or on the toxicity of T. terrestris in fish. An alternate technique for commercial production of all-male fish populations would be to use plant extracts.
The objective of this study is to determine the effect of the extract of T. terrestris on sex reversal in O. niloticus larvae.
1 Results

Table 1 Growth parameters of O. niloticus under different levels of Tribulus terrestris extract in the laboratory

Table 2 Growth parameters of O. niloticus under different levels of Tribulus terrestris extract in the pond

Figure 1 Percentage of male to female
2 Discussion
2.1 Survival Rates and Growth of Fish
Fry and fingerlings in all the treatments of the experiments exhibited high survival rates, though this survival was not uniform, it was observed that fish showed increase in survival rate with increase in T. terrestris level in the feed used in the experiment. This is similar to the observations of Kavitha and Subramanian (2011), and Cek et al., (2007a). This study further showed that T. terrestris has no negative effect on the survival of O. niloticus. The steady weight gain observed during the phase one of this experiment, with increase in T. terrestris inclusion, is similar to the observations of Kavitha and Subramanian (2011), Cek et al. (2007a) and Cek et al. (2007b) on Peocilia latipinna, P. reticulata and Cichlasoma nigrofasciatum respectively. They all reported that fish had better growth rate with increase in the concentration of T. terrestris in culture water. Tribulus terrestris has been reported by (Akram et al., 2011) to be a testosterone enhancer, T. terrestris treated fish showed better growth rates with increase in the levels of T. terrestris. This agrees with the report of Mateen (2007) that since the androgens have both sex reversal and anabolic effects, the sex reversed tilapia shows a better growth performance as compared to untreated tilapia.
2.2 Water Quality Parameters
Temperature ranged between 26 to 28, pH ranged from 7.7 to 8.2, Dissolved oxygen was from 5.13 mg/L to 9.12 mg/L, Nitrite and ammonia both ranged from 0.001 mg/L to 0.044 mg/L and 0.030 mg/L to 0.142 mg/L. Temperature of the pond ranged from 26 to 28, the pH of the pond water was between 7.7 and 7.9, dissolved oxygen ranged from 6.25 mg/L to 9.12 mg/L, Nitirte and Ammonia were between 0.06 mg/L to 0.12 mg/L and 8.71 mg/L to 12.82 mg/L respectively. These water quality parameters of the experimental set up are in conformity with the requirements for adequate growth of O. niloticus as reported by Omitoyin (2007) and Soto-Zarazúa et al. (2010). These water quality parameters are in conformity with the requirements for adequate growth of fish as reported by Omitoyin (2007).
2.3 Sex Reversal Ratio
It is believed that T. terrestris affects androgen metabolism, significantly increasing testosterone or testosterone precursor levels (Neychev and Mitev, 2005) and oral administration of T. terrestris increased sexual behavior in male rats (Gauthaman et al., 2002).
In this study, the inclusion of MT and T. terrestris extract to the fish feed contrary to the addition of T. terrestris in the culture water by Kavitha and Subramanian (2011) and Cek et al. (2007a) showed similar results. As against the usual 1:1 ratio of males to females of O. niloticus, the treatment with 0.0g/kg of T. terrestris extract gave a ratio of 1:2 (males to female); the percentage of males was however, significantly higher than females in all T. terrestris and MT treated fish. The percentage of males recorded in the MT treated fish in this experiment was 73% which is similar to the results obtained by Marjani et al. (2009) at the same concentration for Mozambique tilapia. Kavitha and Subramanian (2011), Cek et al. (2007a; 2007b) and Kavitha et al. (2012) also reported that percentage of males increased with increase in the concentration of T. terrestris in Peocilia latipinna, P. reticulata and Cichlasoma nigrofasciatum. Tribulus terrestris treated 0-day-old larvae showed successful sex reversal and spermatogenesis than untreated progenies.
In the present study, there was a higher recorded percentage of males, growth rates and better feed conversion ratio in the fish treated with T. terrestris at 2.0 g/kg concentration which compares very well with those recorded for the fish treated with MT. It can therefore be concluded that 2.0 g/kg and above concentration of T. terrestris can be used successfully to induce sex reversal in O. niloticus in place of MT which is more expensive, unavailable and has public health issues related to its use.
3 Materials and Method
3.1 Fry Production
Five pairs of broodstocks of Oreochromis niloticus (350±25.8) g were collected from the Department of Aquaculture and Fisheries Management Fish Farm, University of Ibadan and stocked at a ratio of 1:1 (male to female) in 1m X 1m hapas and monitored regularly for fertilized eggs. After 21 days, fertilized eggs were noticed in the mouth of the fish and were collected and incubated in the hatchery for (18-32) hrs. The hatchlings were transferred to the laboratory 24 hrs after hatching.
3.2 Extracts Procurement and Feed Preparation
Tribulus terrestris extract (Trib 60) was procured from Tonvara Premium Natural Supplements, United Kingdom. The extract contained 60% protodioscin, while the 17-α-methyltestosterone which was used as control was purchased from Kingdom Aquarium Nigeria Ltd, Lagos, Nigeria.
A 50% crude protein diet was prepared using soy bean meal, maize, fish meal, vitamins premix, di-calcium phosphate, and salt purchased and milled at Bodija Market, Ibadan. The 50% C.P was based on the protein requirement of O. niloticus fry (30-56) % as recommended by Jauncey (2000). The milled feedstuffs were passed through a 200 μm sieve mesh.
3.3 Experimental Procedures
The experiment was a completely randomized design with six treatments and three replicates respectively representing different inclusion rate of 0.0 g, 1.0 g, 1.5 g, 2.0 g, and 2.5 g T. terrestris extract to 1 kg basal diet (T1-5) treatment 6 containing 50 mg/kg of 17-α-methyltestosterone to basal diet as control. 450 day-old fry ofO. niloticus were randomly distributed into 30L capacity plastic aquaria and were acclimatized for 3 days before the commencement of feeding with experimental diets in the laboratory.
Each aquarium was adequately aerated. The fry were fed with the diet ad libitum from day four thrice daily for 42. Settled fish wastes were siphoned with half of aquarium's water, fresh aerated water was used to replenish daily while the water in the aquaria were changed completely twice a week. At the end of reversal stage, the fish were transferred into the Departmental fish farm into hapas rigged in an earthen pond and were fed for 70 days with commercial diet. The fish were weighed biweekly and water quality parameters monitored. The fish were sexed on the 70th day using a hand binocular. The following growth performance parameters were determined; Mean Weight Gain, Length of fish, Food Conversion Ratio and Specific Growth Rate. Growth performance was determined and feed utilization was calculated according to Ogunji et al. (2000) and Ahmad et al. (2002).
Water quality parameters (temperature, dissolved oxygen, pH, ammonia and nitrite) were determined weekly. Dissolved oxygen, Ammonia and Nitrite were measured using Hach’s Model FF-1A Fish Farmer’s Water Quality Test Kit. Temperature was measured using a mercury-in-glass thermometer. The pH was monitored using Hanna model H1-98107.
Adaikan P. G., Gauthaman K., Prasad R. N., and Ng S. C., 2000, Proerectile pharmacological effects of Tribulus terrestris extract on the rabbit corpus cavernosum, Annals Academic of Medicine Singapore, 29(1), 22–26, PMid:10748960
Ahmad M. H., Shalaby A. M. E. Khattab Y. A. E., and Abdel-Tawwab M., 2002, Effects of 17 α– methyltestosterone on growth performance and some physiological changes of Nile tilapia fingerlings (Oreochromis niloticus L.), Egyptian Journal of Aquatic Biology and Fishieries, 4(4): 295-311
Altun T., Tekelioğlu N., and Danabaş D., 2006, Tilapia Culture and Its Problems in Turkey, E. U. J. Fish. Aquat.Sci., 23(3-4): 473-478
Cek S., Turan F., and Atik E., 2007a, The effects of gokshura, Tribulus terrestris, on sex reversal of guppy, Poecilia reticulate, Pakistan Journal of Biological Science, 10(5): 718-725
Cek S., Turan F., and E. Atik, 2007b, Masculinization of convict cichlid Cichlosoma nigrofasciatum) by immersion in Tribulus terrestris extract, Aquaculture International, 15:109-119
Cek S., Yildirim Y., Sereflisan H., and Akyurt I., 2004, Sex control in aquaculture, In Water Resources Management: Risks and Challenges for the 21st Century eds Harmancıoğlu, O. et. al.), Izmir, Turkey, pp. 343–353
Gale W. l., Fitzpatrick M. S., and Schreck C. B., 1995, Immersion of Nile tilapia Oreochromis niloticus) in’17-α-metyltestosterone and mestanolone for the production of all male populations, in F. Goetz and P. Thomas, eds., the 5th International Symposium on the Reproductive Physiology of Fish, The University of Texas at Austin Printing Department, Austin, TX, USA, pp.117
Ganzera M., Bedir E., and Khan I. A., 2001, Determination of steroidal saponins in Tribulus terrestris by reversed-phase high-performance liquid chromatography and evaporative light scattering detection, Journal of Pharmaceutical Science, 90: 1752–1758
Gauthaman K., Adaikan P. G., and Prasad R. N. V., 2002, Aphrodisiac properties of Tribulus terrestris extract   Protodioscin) in normal and castrated rats, Life Sciences, 71(12): 1385–1396
Gupta M. V., and Acosta B. O., 2004, A review of global tilapia farming practices, Aquaculture Asia, 4(1): 7-16
Gupta M. V., and Acosta B. O., 2004, A review of global tilapia farming practices, Aquaculture Asia, 4(1): 1-16
Jegede T., and Fagbenro O. A., 2007, Tilapia husbandry vs. midwifery: use of antifertility plants, In: Onibi G.E., Agele S.O., Adekunle V.A.J. and Olufayo M.O. (eds.), Proceedings of the Third Annual Conference of School of Agriculture and Agricultural Technology, Federal University of Technology Akure, Nigeria, pp. 118-122
Kavitha P., and Subramanian P., 2011, Effect of Tribulus terrestris on monosex production in Poecilia latipinna, Current Science, 101(1): 100-104
Kavitha P., Ramesh R., and Subramanian P., 2012, Histopathological changes in Poecilia latipinna male gonad due to Tribulus terrestris administration, In Vitro Cellular & Developmental Biology-Animal, 48 (5): 306-312
Lim C. E., and Webster C. D., 2006, Tilapia: biology, culture, and nutrition, The Haworth Press Inc., 10 Alice Street, Bringhamton, NY 13904-1580, USA
Marjani M., Jamili S., Mostafavi P. G., Ramin M., and Mashinchian A. 2009, Influence of 17-alpha methyl testosterone on masculinization and growth in tilapia (Oreochromis mossambicus), Journal of Fisheries and Aquatic Science, 4: 71-74,
Neychev V. K, and Mitev V. I., 2005, The aphrodisiac herb Tribulus terrestris does not influence the androgen production in young men, Journal of Ethnopharmacology, 101(3): 319-323
Ogunji J. O., and Wirth M., 2000, Partial Replacement of Fish Meal with Some Alternative Protein Sources in the Diet of Tilapia (Oreochromis niloticus), Session: Technologies for livestock production and Aquaculture
Omitoyin B. O., 2007, Introduction to fish farming in Nigeria, Ibadan University Press, University of Ibadan, Nigeria, pp.90
Pandian T. J., and Sheela S. G., 1995, Hormonal induction of sex reversal in fish, Aquaculture, 138: 1–22
Penna-Mendoza B., Gomez-Marquez J. L., Salgado-Ugerte I. H., and Ramirez-Nogguera D., 2005, Reproductive biology of Oreochromis niloticus (Perciformes: Cichlidae) at Emiliano Zapata dam, Morelos, Mexico, Revista de biologia tropical., 53: 515–522
Phelps R. P, and Carpenter R. H, 2002, Monosex tilapia production through androgenesis: selection of individuals for sex inheritance characteristics for use in monosex production In: McElwee K., Lewis K., Nidiffer M., and Buitrago P. (Eds.), Nineteenth Annual Technical Report. Pond Dynamics/Aquaculture CRSP, Oregon State University, Corvallis, Oregon, pp.39-44
Soto-Zarazúa G. M, Rico-García E, Ocampo R., Guevara-González R. G., and Herrera-Ruiz G., 2010, Fuzzy-logic-based feeder system for intensive tilapia production (Oreochromis niloticus), Aquaculture International, 18(3):  379-391
Tariq-Ezaz M., Mayers J. M., Powell S. F., McAndrew B. J., and Penman D. J., 2004, Sex ratio in the progeny of androgenic and gynogenetic YY male Nile tilapia, Oreochromis niloticus L., Aquaculture, 232(1–4): 205–214

Wassermann G. J., and Afonso L. O. B., 2003, Sex reversal in Nile tilapia Oreochromis niloticus, (Linnaeus) by androgen immersion, Aquaculture Research, 34(1): 65-71

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