The use of plant extracts to control tilapia reproduction: Current status and future perspectives

Control of prolific reproduction is vital for a profitable tilapia aquaculture enterprise. All-male tilapia culture is a popu-lar method used to control prolific breeding, because the male individuals grow faster than female and mixed-sex populations. Presently, most farmers use 17 α -methyl testosterone (MT) to produce all-male tilapia individuals, although synthetic hormones are linked to human health and environmental risks. Recently, considerable attention has focused on plant-based products as alternatives to MT, because they are affordable, safe, and eco-friendly. Despite the growing interest in using plant extracts to prevent fre-quent spawning in tilapia production, the available information is not collated to standardize application guidelines. Accordingly, this review article consolidates existing knowledge on the use of plant extracts to control prolific breeding in tilapia culture systems. In addition, limitations to commercial application of the extracts are identified. To date, seed, root, and leaf extracts of 20 plant species, most notably, Tribulus terrestris, Mucuna pruriens, and Carica papaya , exhibit potential for controlling unwanted breeding in tilapia production systems. The extracts are mainly administered orally, incorporated in fish feeds. Saponins and flavanoids are the main bioactive compounds in the phytoextracts, which induce sex inversion and fertility impairment in tilapia. The commercialization of plant extracts is, however, hampered by lack of standardized information on extract preparation, optimal dosages, and mechanism of action. Thus, future studies should address these technical limitations and highlight economic incentives for commercial use of plant extracts in tilapia aquaculture. plant extracts' application in controlling unwanted spawning in tilapia aquaculture, as an alternative to the currently, most used synthetic hormones.

which induce sex inversion and fertility impairment in tilapia. The commercialization of plant extracts is, however, hampered by lack of standardized information on extract preparation, optimal dosages, and mechanism of action.
Thus, future studies should address these technical limitations and highlight economic incentives for commercial use of plant extracts in tilapia aquaculture.

K E Y W O R D S
all-male tilapia, aquaculture, phytochemicals, synthetic hormones, 17α-methyl testosterone

| INTRODUCTION
Tilapia is the second most farmed food fish after carps globally. According to the Food and Agriculture Organization of the United Nations (FAO), the global production of tilapia has continued to grow, rising from 3.1 million tonnes in 2010 to 5.6 million tonnes in 2018 (FAO, 2020). This has highlighted the significant contribution of the tilapia industry to the global economy as one of the world's primary sources of proteins for human consumption (FAO, 2020).
The exponential increase in tilapia production is related to the suitable aquacultural attributes: (a) ease to breed in captivity; (b) having a short production cycle because of fast growth rate; (c) acceptability of artificial feeds after yolk-sac absorption; and (d) marketability (El-Sayed, 2006;Mair, 2001). Technology advancements, including control of early maturity and prolific breeding, have also contributed to the expansion of tilapia's global production (Beardmore, Mair, & Lewis, 2001;FAO, 2017;Toguyeni, Fauconncau, Fostier, & Abucay, 2002). Prolific breeding results in excessive fingerling recruitment in grow-out systems causing overpopulation, and competition for resources, and consequently stunted and small-sized fish, which fail to attract good market prices (Teichert-Coddington, Manning, Eya, & Brock, 2000). Therefore, periodic harvesting of fry and fingerlings, high-density culture, cage culture, polyculture with predator fish, sterilization by application of heat shock, and all-male culture, are applied to minimize unwanted reproduction in tilapia production systems (Fortes, 2005;Guerrero, 1982;Mair & Little, 1991). Currently, the culture of all-male tilapia individuals is commonly practiced because, in addition to control of prolific spawning, males grow faster and larger than females, resulting in a shortened production cycle (Baroiller & D'Cotta, ;Beardmore et al., 2001;El-Greisy & El-Gamal, 2012;Megbowon & Mojekwu, 2014).
Various interventions have demonstrated the possibility of utilizing plant extracts as possible options to synthetic steroids in aquaculture in response to the increasing consumer demand for organically produced agricultural products, including fish (Leet et al., 2011). The acceptance of plants for use in aquaculture is linked to ease of access and being relatively safer for the environment and humans, than synthetic hormones (Chakraborty et al., 2013;Logambal, Venkatalakshmi, & Michael, 2000;Olusola, Emikpe, & Olaifa, 2013;Reverter et al., 2014). To date, several plant extracts are predominantly used to improve fish growth, enhance innate immune responses, and control disease in aquaculture, as compared to reproduction control (Baluran, Quiazon, Garcia, Fernando, & Velasco, 2018;Logambal et al., 2000;Olusola et al., 2013;Reverter et al., 2014). Nevertheless, the androgenic compounds present in some plant extracts could be used to control unwanted reproduction in tilapia production systems (Chakraborty et al., 2013;Gabriel et al., 2017;Ghosal, Mukherjee, & Chakraborty, 2021). The phytoandrogens, for example, testosterone, androstenedione, and dehydroepiandrosterone, have been implicated in sex reversal of fish (Godwin, Luckenbach, & Borski, 2003).
In this review, an account of the current state of knowledge on the plant extracts used to control unwanted reproduction in tilapia culture is provided. The literature review strategy involved searching from Web of Science, Science Direct, Google Scholar, and Scopus using "tilapia reproduction control" and "plant extracts" as keywords.
Conference proceedings and doctoral theses were also collected from various libraries using the online catalogue.
The search results were screened and selected by title relevance to the present review, concerning plant extracts for tilapia reproduction control. This search strategy yielded 47 scientific publications, whose findings are reported in this review. The effectiveness of extracts from different plants and the corresponding dosages inducing sex change are documented. Further, the information gaps and the future research directions are highlighted. The review contributes to a better understanding of plant extracts' application in controlling unwanted spawning in tilapia aquaculture, as an alternative to the currently, most used synthetic hormones.

| PLANT EXTRACTS USED IN THE CONTROL OF TILAPIA REPRODUCTION
Plant extracts are becoming an integral part of fish culture, as alternatives to chemicals, drugs, and hormones, in response to the increasing pressure to reduce adverse impacts associated with aquaculture on human and environmental health. The organic plant products are relatively safe, inexpensive and easy to prepare, and are thus viewed as a means to achieving sustainable fish production (Chakraborty et al., 2013;Hoseini, Mirghaed, & Yousef, 2019;Makkar, Francis, & Becker, 2007;Reverter et al., 2014). Moreover, consumers are increasingly demanding good quality and safe fish products, which are free of pollutants (Chakraborty et al., 2013). Therefore, adopting safe and environmentally clean fish production practices will promote tilapia to meet new market requirements. Accordingly, efforts are required to identify and develop novel plant-based products for tilapia production to replace synthetic hormones and chemicals (Ahmed, Fathy, Fayek, & Mohamed, 2019;Citarasu, 2010;Turan & Akyurt, 2005).
The extracts from A. mossambicensis are thus potent in the control of prolific breeding of Nile tilapia.

| Neem tree, Azadirachta indica
A. indica is commonly known as "Neem tree" belongs to order Rutales, and is a member of mahogany family known as Meliaceae (Hashmat, Azad, & Ahmed, 2012). It is a broad-leaved plant that grows up to 30 m tall and with a girth of 2.5 m (Ndodo et al., 2013). The tree is native to the tropical and semi-tropical regions (Silayo & Kiwango, 2010).  (Kapinga et al., 2018). This inconsistency in performance of A. indica extracts could be attributed to differences in extraction media and seasons (Isah, 2019). These discrepancies hamper wide-scale utilization of neem extracts to control tilapia reproduction.
Therefore, more studies are required to clarify the specific effect of H. rosa-sinensis extracts on reproductive organs of fish, before recommendation for commercial adoption as population control agents in tilapia culture systems.

| Soapbark tree, Quillaja saponaria
The soap bark tree belongs to the order Fabales and family Quillajaceae (Guerra & Sepulveda, 2020). This tree is native to China, Peru, and Chile (Angeles Jr., Gallego, Navarro, & Chien, 2017;Francis et al., 2002). The bark of Q. saponaria contains triterpenoidal saponins, mainly triterpene glycoside, which can inhibit fish reproduction and increase growth performance (Bankefors, Nord, & Kenne, 2008;Francis, Makkar, & Becker, 2005;Stadtlander et al., 2008). The feeding of sexually mature female Nile tilapia on Q. saponaria extract at 300 mg kg À1 of diet inhibited spawning (Francis et al., 2005;Francis, Makkar, & Becker, 2001), in addition to inducing masculinization of the female Nile tilapia fry (Table 1). A dose of 700 mg of Q. saponaria saponin kg À1 of diet shifted the sex of Nile tilapia to mostly males (Francis et al., 2002). Similarly, Q. saponaria saponin extracts incorporated in Nile tilapia diets resulted in a significant increase in the number of male individuals (Stadtlander et al., 2008). As such, triterpenoid saponins in Q. saponaria need to be harnessed for production all-male populations in tilapia culture.
In particular, the steroids in M. pruriens extracts increased the serum testosterone in animals (Ahmad, Rahma, Akhtar, & Ali, 2012), stimulating androgenic effects, as was observed in rats (Muthu & Krishnamoorthy, 2011) and fish (Mukherjee et al., 2018). Accordingly, the phytoandrogens in M. pruriens seed extracts produced up to 93% allmale Nile tilapia (Table 1). However, the action of the plant extracts on gonadal function and spawning of tilapia has not been elucidated. Nonetheless, experimental treatment of rats with M. pruriens extract triggered increment in spermatozoa, together with the sexual and androgenic activities (Suresh & Prakash, 2012;Suresh, Prithiviraj, & Prakash, 2009).

| ROUTE OF ADMINISTRATION OF PLANT EXTRACTS
During fish culture operations, plant extracts are administered either through incorporation in fish feeds (oral method), immersion, or injection (Bulfon, Volpatti, & Galeotti, 2015). The oral and immersion techniques are considered noninvasive, hence commonly used methods during reproduction control in tilapia (Table 1). Overall, oral administration is the predominant method because of the low cost of application, potential of treatment of large numbers of fish at the same time, causes no stress or reduced stress (Bulfon et al., 2015;Sakai, 1999;Yoshida, Kruger, & Inglis, 1995) and yields at least 95% sex masculinization (El-Greisy & El-Gamal, 2012;Phelps & Popma, 2000). Nonetheless, the effectiveness of oral method is reduced by differences in the amount of the sex steroids available to the fish, linked to non-uniform distribution of the extracts in the diets during mixing.
The immersion technique is crucial in fish species whose gonadal labile period occurs before first feeding, such as in yolk sac larvae or during embryogenesis (Devlin & Nagahama, 2002). In tilapia, the labile period of sex differentiation occurs during the first 30 days post-hatch, the period within which fry can ingest exogenous diets (Mateen & Ahmed, 2007). Immersion of tilapia in plant extracts during post-hatching stages has yielded up to 90% masculinization (Table 1). However,fish immersion method requires mixing the extracts with culture water at each exposure time , which results in wastages of the masculinization agent, and high rate of release into the environment.
Intra-peritoneal injection of fish is efficient and rapid, necessitated by quick absorption of the extracts (Reverter et al., 2014). However, the process of injecting fish is expensive, laborious, stressful to fish, and impractical for small fish, that is, less than 15 g fish À1 (Anderson, 1992;Beardmore et al., 2001;Blazer, 1992;Reverter et al., 2014;Sakai, 1999). Besides, the method requires technical skills to avoid inflicting damage onto the fish (Hoga, Almeida, & Reyes, 2018). As such, the injection method is rarely utilized to administer sex inversion reversal agents in tilapia culture systems.

| MECHANISM OF ACTION OF PLANT EXTRACTS AS CONTROL AGENTS OF REPRODUCTION IN TILAPIA
The precise mechanisms by which plant bioactive compounds manipulate reproduction in tilapia are not yet elucidated. Nevertheless, two pathways, that is, endocrine system modulation and induction of gonadal histological changes are postulated as plausible functional mechanisms responsible for changes on the reproductive physiology of fish.
The phyto-compounds are hypothesized to inhibit aromatase enzyme Cytochrome P450 aromatase enzyme, which catalyzes the conversion of androgens to estrogens, thus favoring development of female characteristics (Eng et al., 2001). The enzyme is inhibited through either competitive inhibition of natural substrates for the enzyme, decreasing the expression of cyclic adenosine monophosphate (cAMP) responsive element binding (CREB) protein or inhibiting the generation of cAMP. Consequently, the pathway regulating aromatase expression is altered, which in turn augments production of androgens, modifying sex ratio in favor of male individuals (Cheshenko et al., 2008).
Phyto-compounds such as flavonoids and steroidal saponins, are reported to inhibit aromatase enzyme activity (Golan et al., 2008) increasing the production of testosterone, a process towards induction fish masculinization (Gauthaman & Ganesan, 2008).
The phyto-compounds may also antagonize endogenous estrogens by interacting with estrogen nuclear receptors. The antagonicity of phyto-compounds is facilitated by similarity in structures to estrogens and high affinity for estrogen receptors. Besides, the phytochemicals have stable structures and low molecular weights, which permit passage although the cell membranes (Ososki & Kenelly, 2003 (Bennetau-Pelissero et al., 2001;Chen & Chang, 2007;Green & Kelly, 2009;Miyahara et al., 2003;Tarigan et al., 2016). Therefore, the anti-estrogenic compounds can easily bind onto the estrogen receptors, subsequently disrupting the effect of estrogens.

| Induction of gonadal histological changes
The phyto-compounds may impair fertility by inducing histological changes in the gonads of fish: (a) delay gonadal maturation and (b) obstruct reproductive functions. The histological changes in fish testes and ovaries include disintegration of gonad cells, rupture of seminiferous lobule and follicles as well as gonadal necrosis. As a consequence of gonadal damage, testes and ovaries become devoid of spermatids and oocytes, respectively (Abdelhak et al., 2013;Ampofo-Yeboah, 2013;Jegede, 2010;Jegede & Fagbenro, 2008a, 2008b. For example, the saponins in C. papaya seed powder rendered the testes and ovaries of O. niloticus and O. mossambicus devoid of spermatids and oocytes (Ampofo-Yeboah, 2013;Jegede & Fagbenro, 2008a, 2008bSolomon et al., 2017).

| LIMITATIONS TO THE UTILIZATION OF PLANT EXTRACTS AND FUTURE PERSPECTIVES
The use of plant extracts to control prolific spawning in tilapia culture systems is an emerging innovation. As such, a number of drawbacks continue to constrain commercial application of phytoextracts. Generally, technical and economic limitations continue to hinder the progress in application of plant extracts from experimental to field levels (Chakraborty et al., 2013;Ghosal & Chakraborty, 2020;Kapinga et al., 2019;Mukherjee et al., 2018).

| Technical limitations
The existing knowledge lack reliable information on the: (a) ideal extraction methods of phytochemicals, (b) effective bioactive compounds in plants, (c) effect of seasons, environmental parameters and stage of plant growth on the yield and biological activity of extracts, (d) optimal dose requirements, (e) precise mechanism of action, and (f) longterm effects of plant extracts on quality of fish's flesh and physiological processes such as growth and immunity (Chakraborty et al., 2013;Gabriel et al., 2017;Ghosal et al., 2021;Mukherjee et al., 2015b;Stadtlander et al., 2008).
The present review observed discrepancies in extraction methods of plant extracts, especially extraction solvents used, yet the yield and functional properties of phytochemicals are dependent on the extraction techniques (Dhanani, Shah, Gajbhiye, & Kumar, 2017;Tiwari, Kumar, Kaur, Kaur, & Kaur, 2011). A variety of bioactive compounds from different plant extracts have varying solubility properties in different solvents such as aqueous, ethanol and methanol (Truong et al., 2019). It is therefore necessary to select an ideal extraction solvent especially, of appropriate polarity, to maximize the yield of the target bioactive compounds without disrupting their functional activities (Dhanani et al., 2017). Further, little is known on the effect of the plant's growth stage (Akula & Ravishankar, 2011), seasons and environmental parameters (Isah, 2019) and geographical origin (Dinchev et al., 2008), on the concentration and composition of phytochemicals believed to control reproduction in tilapia. With regard to the effectiveness of plant extracts, variable optimal doses are reported by different studies, even for extracts from the same plant (Table 1). Similarly, the effects of specific bioactive compounds from the same plant are not defined. The reviewed studies, therefore, attribute the observed effects of plant extracts to various phytochemicals present in the plant. In addition, the precise mechanism modulating tilapia sex inversion or fertility impairment is not understood, further hampering the harmonization of the optimal dosages and treatment regimen.
Given the technical information gaps in utilizing plant extracts in tilapia culture, low reproducibility of results is reported, a concern that needs to be addressed before commercialization of the technology. Research on developing efficient methods for isolating and quantifying bioactive compounds in plant extracts, including determining ideal extraction solvents for target phytochemicals that induce sex manipulation in tilapia is vital. Further, the hypothesized mechanisms of action necessitate comprehensive analysis either by searching for sex gene expression profiles at the transcriptomic level or changes in the sex steroid levels (Capel & Tanaka, 2013;Ross & Capel, 2005), to validate the specific role/s of plant extracts on the tilapia sex differentiation pathway. This would facilitate the designing of a standard utilization protocol including: extraction procedure for bioactive ingredients of interest, extraction solvents that result in highly purified concentrations, administration approaches, and subsequently optimal doses levels of plant extracts (Dhanani et al., 2017;Mukherjee, Ghosal, Moniruzzaman, De, & Chakraborty, 2019).
The commercial application of plant extracts in masculinization of tilapia is also limited by inadequate information regarding the long-term effects of the extracts on the quality of fish's flesh and other physiological processes.
Plant extracts may either positively or negatively modulate the physiological functioning of fish, depending on plant type and the administered dose. Besides reproduction control, plant extracts such as saponins also enhance the immune system and subsequently growth performance of fish. However, a specific dosage of the extracts that is effective in controlling unwanted breeding may trigger negative growth response in the same fish. Some plant extracts also contain antinutritional factors such as gossypol in cotton, which interfere with feed utilization, hence adversely affecting health and growth of fish. (Ayotunde & Ofem, 2008;Chakraborty et al., 2013;Gabriel et al., 2015;Kapinga et al., 2018;Prasad & Mukthiraj, 2011). Thus, in-depth explorations focusing on determining optimal doses of plant extracts for sex manipulation that do not cause undesirable effects to the fish's flesh and physiological processes are needed.

| Economic limitations
Although viewed as relatively cheap and readily available alternative to synthetic hormones, no study has sufficiently described the economic viability of utilizing plant extracts in aquaculture. As such, information on the cost effectiveness of plant-based products for commercial application in tilapia culture is lacking. Therefore, research to determine the benefit-cost ratio of utilizing plant extracts is needed to guide recommendations for an economically feasible alternative to synthetic hormones.

| CONCLUSION
This review underpins the potential of plant extracts as alternatives to synthetic steroids in controlling unwanted reproduction in tilapia culture. However, addressing the technical limitations and generating information on economic incentives are key to fostering adoption of plant extracts as effective, environmentally sustainable, and socially acceptable approach to masculinization of tilapia.

CONFLICT OF INTEREST
The authors of this study declare no conflict of interest.