Application of phytochemicals as growth-promotersand endocrine modulators in fish cultureSuman B. Chakraborty1,2, P�eter Horn1 and Csaba Hancz1

1 Faculty of Animal Science, Kaposv�ar University, Kaposv�ar, Hungary

2 Department of Zoology, Serampore College, Serampore, West Bengal, India

Correspondence

Suman B. Chakraborty, Faculty of Animal

Science, Department of Nature Conservation,

Kaposv�ar University, H-7401 Kaposv�ar, PO Box

16, Hungary. E-mail: sumanbc76@gmail.com

Received 24 July 2012; accepted 27 November

2012.

Abstract

There is a constant need to increase productivity in aquaculture, particularly to

improve growth rate, feed utilization as well as stress resistance of fish. Because of

consumer concerns and strict regulations in many countries, the use of synthetic

chemicals, hormones and antibiotics is becoming unviable and natural com-

pounds are more acceptable to the public. A wide variety of chemical compounds

are found in plants, and many of them have been shown to have beneficial effects

on appetite, growth and the immune status of fish acting through different mech-

anisms. Phytochemicals contained in herbs may enhance the innate immune sys-

tem, possess antimicrobial capabilities, and are redox active molecules with

antioxidant characteristics that may help to improve the general physiological

condition of fish. Many studies have discussed the values of phytochemicals as

feed additives. Another paramount concern related to phytochemicals is their

endocrine modulator effect that can be applied both in aquaculture targeting the

production of table fish and the growing sector of ornamental fish production.

Different mechanisms such as the effects at the steroid receptor level, effects on

steroid synthesis, distribution and excretion, actions on the hypothalamus–pituitary–gonad axis, as well as indirect mechanisms including thyroid and

growth hormone disruption have been postulated for the reproductive endocrine

disruption in fish populations by phytochemicals. This paper reviews the results

of a great number of studies focusing on phytochemicals such as essential oils,

saponins, flavonoids and phytosterols discussing their effects on productive traits

and the putative mechanism of action.

Key words: endocrine modulators, fish culture, growth promotion, in vivo application, mecha-

nism of action, phytochemicals.

Introduction

Fish is a source of high-quality protein, vitamin D, sele-

nium, omega-3 fatty acids and other nutrients (Shim et al.

2009). Consumer demand for fish products is increasing

constantly, while wild fish stocks are rapidly declining,

mainly because of overfishing. According to the Food and

Agriculture Organization of the United Nations (FAO

2007), 47% of global fish stocks are fully exploited, thus

offering no reasonable expectations of further expansion,

and another 18% are reported as over exploited. Sustaining

fish supplies from capture fisheries will, therefore, not be

able to meet the growing global demand for fish products.

On the other hand, fish culture has become an important

industry and is the world’s fastest growing sector of agricul-

tural business (Villa-Cruz et al. 2009). Globally, fish culture

is expanding into new directions, intensifying and diversi-

fying. Several growth promoters and hormones have been

tested for enhancing feed conversion efficiency and for

increasing fish culture productivity (Makkar et al. 2007).

However, the recent consumer demand for farmed fish has

increasingly stressed quality and safety, and the absence of

pollutants, antibiotics and carcinogens. Thus, along with

growth performance, the fish rearing strategy needs to

focus on food hygiene. Since the European Union ratified a

ban in 2006 for the use of all sub-therapeutic antibiotics

(Regulation 1831/2003/EC), scientists have intensified

efforts to identify and develop safe dietary supplements and

additives that enhance the life activity, health and immune

system of farm fish (Ji et al. 2007b; Shim et al. 2009).

© 2013 Wiley Publishing Asia Pty Ltd 1

Reviews in Aquaculture (2013) 5, 1–19 doi: 10.1111/raq.12021

Phytochemicals are a large group of plant-derived

compounds that are commonly found in fruits, vegetables,

beans, cereals and plant-based beverages such as tea and

wine (Arts & Hollman 2005). Based on their chemical

structure, phytochemicals can principally be categorized

into alkaloids, flavonoids, pigments, phenolics, terpenoids,

steroids and essential oils. Phytochemicals have been

reported to enhance various activities such as growth, feed

consumption, act as a tonic in immunostimulation, anti-

stress and to promote antimicrobial properties of fish

(Citarasu 2010; Chakraborty & Hancz 2011). Phytochemi-

cals, in the form of herbal biomedicine, has a long history,

mainly in Asian countries (Ji et al. 2009). An overview of

the major potential health benefits of some phytochemicals

is presented in Table 1. These might provide a useful

source of new medicines, pharmaceutical entities and

bioactive compounds for enhancing fish production and

health; and food safety and quality, while conserving the

aquatic environment. Recent research has begun to demon-

strate positive impacts of the application of phytochemicals

and herbal products in fish culture (Rawling et al. 2009;

Chakraborty & Hancz 2011). Intensive efforts have been

made in exploiting plants, plant extracts or natural plant

compounds as potential natural alternatives for enhancing

fish productivity. The objective of this paper is to review

the current research application of phytochemicals or

herbal extracts in finfish culture.

Phytochemicals as Growth Promoting Agents

One of the primary concerns of fish culture is to provide a

diet containing all the necessary nutrients in suitable pro-

Table 1 Potential health benefits of some phytochemical compounds

Phytochemical Source Possible benefit References

Isoflavones Soybeans, soy milk Antioxidant activity, improvements of

growth and serum biochemical attributes,

reduction in blood pressure, increased

vessel dilation

Arora et al. (1998); Erdman et al.

(2007); Lien et al. (2009)

Anthocyanins Blueberries,

strawberries, red

wine

Inhibition of nitric oxide production,

improvement of vision, induction of

apoptosis, decreased platelet aggregation,

neuroprotective effect, anti-diabetic effect,

antioxidant activity, anti-allergic, anti-

inflammatory, anti-microbial activity

Erdman et al. (2007); Ghosh and

Konishi (2007)

Terpenes Saffron, ginger,

cinnamon, coriander,

turmeric

Antioxidant activity, alteration of

biotransformation enzyme activity,

immunostimulation, antimicrobial activity

Lampe (2003); Citarasu et al. (2006);

Rattanachaikunsopon and

Phumkhachorn (2009); Chakraborty

and Hancz (2011); Pandey et al.

(2012)

Proanthocyanidins,

flavan-3-ols

Grapes, red wine,

cocoa

Antioxidant activity, inhibition of LDL

oxidation, inhibition of cellular oxygenases,

chemoprevention of cellular damage,

inhibition of proinflammatory responses in

the arterial wall

Joshi et al. (2001); Erdman et al.

(2007)

Sulfides, thiols Garlic, onion, olives Decrease in LDL cholesterol,

immunostimulation, antimicrobial activity

Dinkova-Kostova (2008);

Chakraborty

and Hancz (2011); Pandey et al.

(2012)

Phenylproponoids,

cinnamic acid,

euganol

Cinnamon, cloves Antioxidant activity, alteration of

biotransformation enzyme activity,

immunostimulation, antimicrobial activity

Lampe (2003); Chakraborty and

Hancz (2011); Subeena and Navaraj

(2012)

Diarylheptanoids,

curcumin

Turmeric Antioxidant activity, scavenging of free

radicals

Lampe (2003); Chakraborty and

Hancz (2011)

Carotenoids Carrots, tomatoes,

various fruits,

vegetables

Neutralization of cell damaging free

radicals,

antioxidant activity

Dutta et al. (2005); Dinkova-Kostova

(2008)

Isothiocyanates Broccoli, mustard

seed

Neutralization of cell damaging free

radicals,

protection against some cancer

Dinkova-Kostova (2008); Juge et al.

(2007)

Alkaloids Tea, coffee, cocoa Antioxidant activity, anti-angiogenesis,

improvement of cardiac health

Hesse (2002); Citarasu et al. (2006);

Chakraborty and Hancz (2011)

Reviews in Aquaculture (2013) 5, 1–19

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S. B. Chakraborty et al.

portions to optimize the growth performance of fish, while

reducing the cost of production. Increasing demand, an

unstable supply and the high price of fishmeal made it nec-

essary to evaluate alternative protein sources of plant origin

in fish diets as partial or total replacement for fishmeal

(Gatlin et al. 2007). But the presence of antinutritional fac-

tors in plant products can affect the utilization of food by

fish and thus limit their application in fish feed (Glencross

et al. 2006). However, there is a changing perception

regarding the potential of various plant secondary metabo-

lites and various processing techniques attempted to elimi-

nate the antimetabolic factors and toxic principles of plant

protein sources in fish feed, have resulted in partial success.

Some studies have been done in which medicinal herbs

containing different phytochemicals, as growth promoting

dietary additives, were fed to fish (Table 2).

Although growth-promoting synthetic hormones have

never been used commercially in aquaculture, recently

implemented strict regulations of food safety are prompt-

ing investigators to seek alternate applications of plant

derived agents (Turan & Akyurt 2005a).

Essential oils

Essential oils are concentrated hydrophobic liquid com-

pounds characterized by a strong odour and are formed by

aromatic plants as secondary metabolites. They contain a

variety of volatile molecules such as terpenes and terpenoids,

phenol-derived aromatic components and aliphatic compo-

nents, and are used as antimicrobial, analgestic, sedative,

anti-inflammatory, spasmolytic and locally anaesthetic rem-

edies. These are very complex natural mixtures containing

quite different concentrations of 20–60 components, charac-

terized by fairly high concentrations of two or three major

compounds compared with trace amounts of other compo-

nents (Bakkali et al. 2008). Some of the essential oils have

been reported to constitute effective alternatives or as

complements to synthetic compounds of the chemical

industry for application in human health, agriculture and

the environment (Carson & Riley 2003).

A commercial product with oregano essential oil

extracted from Origanum heracleoticum, containing monot-

erpenoid phenolic compounds carvacrol and thymol as

major phytochemicals, was shown to act as a growth pro-

moter in channel catfish, Ictalurus punctatus (Zheng et al.

2009). The fish were fed diets containing either carvacrol

extract (0.05%), thymol extract (0.05%), mixture of carvacrol

and thymol extracts (0.0485% and 0.0015%, respectively)

or the commercial product (0.05%) for 8 weeks. Channel

catfish fed with the commercial diet showed significantly

higher (P < 0.05) weight gain, protein efficiency ratio

(PER), condition factor (CF) and improved feed conver-

sion ratio (FCR) compared with fish fed with other diets.

The authors speculated that the reason for this effect might

be linked to the known antimicrobial properties of oregano

essential oil that could control the gut microflora and posi-

tively influence fish performance. In addition to thymol

and carvacrol, oregano essential oils have been reported to

contain more than 30 different active minor constituents

(including two monoterpene hydrocarbons, c-terpineneand q-cymene) (Zheng et al. 2009). A synergism between

all the compounds may have played an important role in

improving the growth performance of fish fed a diet

containing the commercial product compared with the fish

fed diets with thymol and carvacrol alone or in combina-

tion. Such synergism between carvacrol and cymene was

also observed during an in vitro study against drug resistant

Salmonella typhi (Rattanachaikunsopon & Phumkhachorn

2009). The use of phytochemicals in combination rather

than alone may enhance the non-specific, specific immu-

nity and disease resistance in fish. An extensive study

regarding the mechanism of action of each of the

compounds may help to explain the synergism between the

compounds.

Saponins

Saponins are surface active sterols or triterpene glycoside

compounds found in a variety of plants. This group of

plant secondary metabolites derives its name from its ability

to form stable, soap-like foams in aqueous solutions and is

reported to possess diverse properties, beneficial to health

(Shi et al. 2004). These molecules are reported to have con-

siderable commercial value and may be exploited as drugs,

medicines and adjuvants. Saponins are present in tradi-

tional medicine preparations (Xu et al. 1996; Nassiri &

Hosseinzadeh 2008) and have been suggested to affect the

immune system in ways that protect the human body

against cancers (Haridas et al. 2001). In spite of being toxic

to cold-blooded organisms including fish at particular

concentrations, saponin-rich plants may have potential for

exploitation in fish production systems (Makkar et al.

2007).

The dietary ginseng herb containing saponin triterpenoid

glycosides called ginsenosides (or panaxosides) as active

chemical components greatly enhanced the growth, diet

utilization efficiency and haematological indices in Nile

tilapia, Oreochromis niloticus, fingerlings (Goda 2008). The

fundamental skeleton of the genuine sapogenins is

dammarane-type tetracyclic triterpene, and more than 25

such saponins have been identified as the characteristic

principles of white and red ginseng (Shibata 2001). Nile

tilapia fed diets containing either 50, 100, 150, 200 or

250 mg kg�1 of the herb for 17 weeks showed significantly

higher (P � 0.05) growth performance compared with

fish fed a diet with no herbal supplementation. Red blood

Reviews in Aquaculture (2013) 5, 1–19

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Phytochemicals in fish culture

cell counts, haematocrit, haemoglobin, total plasma protein

and total plasma globulin levels significantly (P � 0.05)

increased with increasing dietary herbal levels compared

with those of the control diet fed fish. The herb may reduce

stress, stimulate the immune system, increase protein syn-

thesis and thus may confer a growth increase in fish. Fur-

ther study is required to isolate, characterize and find the

effect of the principal active compound in ginseng on fish

growth in order to speculate that the increase in growth is

induced by the saponin or by the synergistic action of many

other constituents present in the herb.

Quillaja saponin (QS), a triterpene glycoside compound

derived from Quillaja saponaria, has been reported to have

the potential to increase growth in culture fish species,

Table 2 Dietary administration of herbal phytochemicals as growth promoters in fish culture

Plant Phytochemicals Fish species Dose and length of

administration

Results References

Mixture of Massa

medicata fruits,

Crataegi fructus

fruits, Artemisia

capillaries

leaves,

Cnidium

officinale root

Carotinoids, flavonoides,

cinnamic acids, benzoic acids,

folic acid, ascorbic acid,

tocopherols, tocotorienols

Pargus major

juveniles

(mean body weight

24.0 � 0.2 g)

0.5% – 12 weeks Weight (+35%)

FE (+27%)

SGR (+34%)

Ji et al. (2007b)

Artemisia cina

Matricaria

chamomilla

Santonin, betaine, choline,

tannins, pigments, essential oil

Essential oil

Clarias gariepinus

fingerlings (body

weight 22.0 g, total

length 12 cm)

5.0% – 1 month

1.0% – 1 month

NWG (+10%)

FCR (�47%)

NWG (+10%)

FCR (�47%)

Abdelhadi et al.

(2010)

Crude methanol

extract of

Withania

somnifera leaves

Crude methanol

extract of

Ocimum

sanctum

leaves

Alkaloids, withaferin-A,

withanine, somniferine,

somnine, somniferinine,

withananine, pseudo-

withanine,

tropine, pseudo-tropine,

cuscohygrine, anferine,

anhydrine, steroidal lactones

Oleanolic acid, ursolic acid,

rosmarinic acid, eugenol,

carvacrol, linalool, b-

caryophyllene

Epinephelus tauvina

juveniles (body

weight

30.0 � 0.5 g)

100 mg kg�1 – 12 weeks

200 mg kg�1 –

12 weeks

Weight gain (+40%)

FCR (+15%)

SGR (+30%)

Weight gain

(+30%)

FCR (+11%)

SGR (+22%)

Sivaram et al.

(2004)

Phyllanthus niruri

dried powder

Aloe vera inner

gel

Flavonoids, alkaloids,

terpenoids,

lignans, polyphenols, tannins,

coumarins, saponins

Aloin, glucomannans, salicylic

acid

Carassius auratus

adults (body weight

3.481–3.693 g)

1.5% – 60 days

1.0% – 60 days

DWG (+21%)

SGR (+18%)

SGR (+1%)

Ahilan et al.

(2010)

Trifolium

pretense

extract

Genistein Oreochromis aureus

fingerlings (body

weight 0.31 g)

100 mg kg�1 – 90 days SGR (+6%)

FCR (�28%)

PER (+12%)

Turan (2006)

Rheum officinale Anthraquinone derivatives,

emodin, rhein, chrysophanol

Cyprinus carpio var.

jian fingerlings

(body

weight 5.39 g)

2.0% – 10 weeks SGR (+9%)

FCR (�23%)

Xie et al. (2008)

Cynodon

dactylon

ethanol extract

Flavonoids, sterols Catla catla adults

(body weight

88.05 g)

5.0% – 45 days SGR (+40%)

FCR (+25%)

Protein content

(+7%)

Kaleeswaran

et al.

(2010)

‘+’ symbol represents an increase and ‘�’ symbol represents a decrease in the specified response for the best treatment regime with respect to the

control value. Value in brackets indicates the percentage difference between the best treatment regime and the control value for the specified

response.

FE, feed efficiency; SGR, specific growth rate; NWG, net weight gain; FCR, feed conversion ratio; DWG, daily weight gain; PER, protein efficiency

ratio.

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S. B. Chakraborty et al.

reduce their metabolic rate and to suppress reproduction

(Francis et al. 2005). The main aglycone moiety of QS is

quillaic acid (a triterpene of predominantly 30-carbon

atoms of the Δ12-oleanane type), which is substituted with

a di- or trisaccharide at C-3 (X1) and an oligosaccharide at

C-28 with an X2-, X3- and X4-substituted fucose as the first

monomer (San Mart�ın & Briones 1999). Nile tilapias were

given diets supplemented with Quillaja saponin at concen-

trations of 150 and 300 mg kg�1 diet for 14 weeks (Francis

et al. 2001). While fish fed with 150 mg kg�1 diet had a

higher growth rate for the initial 3 weeks of feeding with

the experimental diet, at the end of 14 weeks the group

given Quillaja saponin supplemented diet at a level of

300 mg kg�1 diet showed the highest weight gain, energy

retention, apparent lipid conversion, carcass fat and energy

content. The authors speculated that the less pronounced

growth promoting effect of QS at a higher concentration

during the initial experimental period probably occurred

because the higher QS dose caused excessive damage to the

intestinal mucosa; while the effect of dietary saponins

seemed to decrease with progress in the experiment, proba-

bly due to adaptation by the fish, and growth rate in the

higher QS concentration group increased during the

remaining experimental period. In another experiment,

Francis et al. (2002b) observed that the absolute increase in

weight was higher in QS fed Nile tilapia compared with

control fish, even at a higher dietary level of 700 mg kg�1.

Interestingly, during experiments in which common

carp, Cyprinus carpio, were fed QS supplemented diets at

concentrations of 150 and 300 mg kg�1 diet for 14 weeks,

the growth-promoting effects of QS were most pronounced

at 150 mg kg�1 diet (Francis et al. 2002c). The weight gain,

average protein utilization values and average energy reten-

tion value at the end of the experiment were the highest in

fish fed QS supplemented diet at a concentration of

150 mg kg�1. This group showed significantly better

(P < 0.05) average FCR and metabolic growth rate values

than the control group for up to 4 weeks, but the differ-

ences narrowed in time and were no longer significant

(P > 0.05) at the end of the experiment. The average meta-

bolic rate and oxygen consumption per unit body weight

showed no significant difference (P > 0.05) among the

groups. However, the consumption of QS containing diets

during alternate weeks did not result in retention of the

more pronounced initial growth-promoting effects during

the entire experimental period in carp (Francis et al.

2002d). Such variations in the optimum treatment method

in different fish species may be related to the physiological

differences among the species. The mechanisms contributing

to growth promoting effects of QS remain unclear. Quillaja

saponin may increase the activity of the enzymes amylase

and trypsin, suggesting stimulation of protein and carbohy-

drate digestion in the intestine (Francis et al. 2002a). An

increase in liver enzymes, lactate dehydrogenase (LDH)

and cytochrome c-oxidase (CO) has also been observed on

feeding a QS containing diet (Makkar et al. 2007). Thus, it

may be suggested that Quillaja saponin promotes the respi-

ratory chain pathway and enhances energy availability,

thereby causing growth increase. Considering these results,

QS may be argued to be one of the most potent plant-

derived growth promoting agents in fish, but further stud-

ies are required to design optimum inclusion levels of sapo-

nins, determination of the treatment regime and methods,

and its functional mechanism in a species specific manner.

The addition of Gynostemma pentaphyllum, a traditional

Chinese herbal medicine containing triterpenoid saponins

(gypenosides), to grass carp, Ctenopharyngodon idella, feed

resulted in a lower FCR and higher specific growth rate

(SGR) (Wu et al. 1998). The gypenosides are structurally

related to ginsenosides and this oriental herb has been

known to possess adaptogen and antioxidant properties

(Liu et al. 2005). But, whether this growth promoting effect

of the herb is due to the saponin or synergistic action of

different constituents remains unclear.

Soysaponins are a group of complex and structural

diverse oleanane triterpenoids with sugar moieties attached

at positions C-3 (group A, bidesmosidic) and C-22 (group B,

monodesmosidic) of the ring structures (Zhang & Popo-

vich 2009). In two feeding trials, Twibell and Wilson

(2004) analysed the effects of dietary soybean meal (SBM),

purified soybean saponin and supplemental cholesterol

concentrations on growth responses of juvenile channel

catfish and found no significant difference (P > 0.05) in

weight gain and feed intake between fish fed a control diet

without SBM and fish fed a diet containing purified soy-

bean saponin. Analysing the results, the authors opined

that soy saponin was not responsible for reduced feed con-

sumption in fish fed high dietary SBM concentrations, and

supplemental cholesterol might improve the growth

response in juvenile channel catfish fed SBM-based diets. A

careful monitoring in this regard is necessary to determine

the concentration of soy saponin that may be applied in the

feed of different fish species.

Protodioscin, a steroidal saponin compound, has been

reported to be the putative active component of the herb

Tribulus terrestris (Dinchev et al. 2008). The growth pro-

moting effects of T. terrestris extract were recorded during

immersion experiments on convict cichlid, Cichlisoma

nigrofasciatum (C�ek et al. 2007a) and guppy, Poecilia reticu-

lata (C�ek et al. 2007b), and T. terrestris extract treated fish

exhibited successful growth acceleration and significantly

(P < 0.05) increased growth rate compared with the

control group. Turan and C�ek (2007) observed the largest

gain in body weight (P < 0.05) of African catfish, Clarias

gariepinus at the end of a 30 days immersion treatment

(thrice weekly) with 9 g 30 l�1 T. terrestris extract.

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Phytochemicals in fish culture

Protodioscin has been demonstrated to increase the levels

of different androgens in mammals (Gauthaman &

Ganesan 2008) and these steroidal hormones may have ren-

dered anabolic affects leading to a growth increase in fish as

well. However, the authors did not measure the plasma tes-

tosterone levels during the experiments, and hence could

not provide any conclusive evidence in this regard.

Flavonoids

Flavonoids are diphenylpropanes that constitute one of the

most characteristic classes of secondary metabolites in

plants (Cao et al. 1997). The structural components com-

mon to these molecules include two benzene rings on either

side of a 3-carbon ring, and multiple combinations of

hydroxyl groups, sugars, oxygens and methyl groups

attached to these structures create the various classes of

flavonoids: flavanols, flavanones, flavones, flavan-3-ols

(catechins), anthocyanins and isoflavones. Flavonoids have

been shown to be potent antioxidants, capable of scavenging

hydroxyl radicals, superoxide anions and lipid peroxy

radicals, and have been reported as having antibacterial,

anti-inflammatory, antiallergic, antimutagenic, antiviral,

antineoplastic, anti-thrombotic and vasodilatory actions

(Yao et al. 2004; Chakraborty & Hancz 2011).

Isoflavones such as genistein and daidzein are often

termed phytoestrogens due to their structural resemblance

with 17b-oestradiol and oestrogenic and/or antioestrogenic

properties. However, the functional group of phytoestro-

gens includes other naturally occurring substituted poly-

phenolic nonsteroidal plant bioactive compounds such as

coumestans, lignans and resorcylic acid lactones (Turan

2006). These compounds concentrated in various food

sources such as soy and other legumes, berries, fruits, vege-

tables, nuts, broccoli and sprouts have been reported to

exhibit vital biological functions for human health and

received increasing attention because of their biological

properties and pharmacological role in the prevention of

human cancer and other diseases (Ziegler 2004). Attempts

have been made to use the therapeutic potential of phytoes-

trogens, containing principally isoflavones, for growth

induction in finfish culture, as well.

Genistein (4′,5,7-trihydroxyisoflavone), a weak oestro-

genic flavonoid found in soybean products, has a diphenol

structure that resembles stereochemically human endoge-

nous 17b-estradiol (Tham et al. 1998; Cassidy 1999).

Ko et al. (1999) reported the effect of genistein on the

growth and reproductive development of yellow perch,

Perca flavescens. Fish were fed diets containing genistein at

concentrations of 7.5 and 0.75 mg g�1 diet, and 17b-oes-tradiol at a concentration of 10 lg g�1 diet. The oestradiol

diet promoted weight gain in yellow perch of both sexes.

The genistein diet at a concentration of 7.5 mg g�1 diet

decreased weight gain in females, while the growth of the

fish fed diet containing genistein at a concentration of

0.75 mg g�1 diet showed no difference in growth of either

the estradiol fed fish or the control diet fed fish. Based on

their results, the authors suggested that genistein might

have a positive effect on growth in yellow perch. However,

more research is warranted in order to explain such dose

dependent and sex specific variations in fish growth after

genistein treatment.

Turan and Akyurt (2005b) reported results from an exper-

iment evaluating the effects of a red clover, Trifolium pre-

tense, extract, containing high levels of oestrogenic

isoflavone genistein, on growth and body composition of

C. gariepinus. The fish were fed with diets containing red

clover extract at concentrations of 25, 50 and 75 mg kg�1

diet for 120 days and the 75 mg kg�1 diet group showed the

highest final weight, weight gain, SGR, protein and lipid

contents. The presence of phytoestrogen in red clover extract

may enhance nutrient utilization and stimulate growth hor-

mones leading to an increase in the body growth of fish. In a

mammalian model, dietary supplementation with red clover

extract resulted in increased plasma concentrations of

growth hormone (GH) and insulin-like growth factor

(IGF)-I, suggesting a physiological mechanism for the

increased growth rates of those animals (Moorby et al. 2004)

and a similar mechanismmight be postulated also in fish.

In an immersion experiment with a commercial mixture

of phytoestrogens, Yılmaz et al. (2009) found that the SGR

and protein content of female C. gariepinus increased with

increasing rates of the phytoestrogen mixture up to a con-

centration level of 420 mg 30 L�1 (P < 0.05), but there was

no significant affect of the mixture on the growth perfor-

mance of males (P > 0.05). Interestingly, treatment with

higher concentrations of the mixture resulted in compara-

tively lower growth in the fish suggesting an optimum level

of treatment with the phytoestrogens. However, the reasons

for such a sex-specific growth increase and reduced growth

potential at higher concentrations of the mixture have not

been elucidated in the study. In fact, variability of results

for phytochemical treatments in not only different fish

species, but also in different sexes and at different life-cycle

stages within the same species may limit the application of

phytoconstituents in finfish culture. Finding the functional

mechanisms behind such diverse effects of phytochemicals

should be an important field of research.

The dietary administration of green tea, Camellia

sinensis, leaves that contain flavonoid catechins such as epi-

catechin, epicatechin gallate, epigallocatechin and epigallo-

catechin gallate as bioactive principles, at a concentration

of 0.5 g kg�1 diet for 12 weeks enhanced the growth, FCR

and protein content of Nile tilapia (Abdel-Tawwab et al.

2010). Catechins possess two benzene rings and a dihydro-

pyran heterocycle with a hydroxyl group on C-3 as the

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S. B. Chakraborty et al.

basic structure, and have been reported to be powerful

antioxidants and to enhance non-specific humoral and

cellular immune system functions in fish (Harikrishnan

et al. 2011a), and hence may stimulate general health

conditions and growth increase in fish, as well.

Cho et al. (2007) evaluated the effect of a 5% dietary

inclusion of various sources (raw leaves, dry leaves,

by-product and extract) of green tea on the growth, body

composition and blood chemistry of juvenile olive floun-

der, Paralichthys olivaceus. At the end of the 7-week feeding

trial, fish fed the control diet and the experimental diet

containing green tea extract showed improved weight gain,

SGR, PER and FCR compared with fish fed other experi-

mental diets. However, all sources of green tea were effec-

tive in lowering serum low-density lipoprotein cholesterol

and glutamic oxaloacetic transaminase concentration. The

authors speculated that the poorer growth of fish fed diets

containing raw or dry leaves or the by-product of green tea

was due to the low consumption of these diets by the fish

because of high fibre content in those diets. Thus, the most

effective doses, application methods and administration

regimes need to be investigated and confirmed before the

application of green tea in finfish culture.

Alkaloids

Alkaloids are heterocyclic organic compounds of plant

origin, normally with basic chemical properties and con-

taining nitrogen in a negative oxidation state (Hesse 2002).

Many alkaloids exhibit marked pharmacological activity,

and have physiological effects that render them valuable as

medicines. Isoquinoline alkaloids represent one of the larg-

est and most interesting groups of plant secondary metabo-

lites as potential alternative growth promoters (Faddejeva

& Belyaeva 1997).

Red tilapia, O. niloticus, fed diets containing a commer-

cial product with the isoquinoline alkaloid sanguinarine, at

inclusion levels of 25, 50, 75 and 100 mg kg�1 for 60 days,

showed significant (P < 0.05) elevations in the mean daily

feed intake, weight gain, SGR and total leukocyte levels.

But other haematological parameters such as haematocrit,

erythrocyte count, haemoglobin, serum glucose level, and

hepatic alkaline aminotransferase activity and hepatoso-

matic index remained unaffected (Rawling et al. 2009).

Sanguinarine belonging to a group called benzo[c]phenan-

thridine alkaloids (QBA), has been reported to display a

number of useful medicinal properties including antimi-

crobial, anti-inflammatory and immunomodulatory

(Yao et al. 2010), and to promote animal growth by

increasing feed intake and decreasing amino acid degrada-

tion from decarboxylation (Kosina et al. 2004). However,

the increase in growth was not dose dependent and thus a

definite conclusion regarding the inclusion level of the

product in fish diet could not be drawn from the study.

Further research is oriented with different fish species and

longer time scales to evaluate its application at industrial

farming levels.

Triterpenoids

Triterpenoids are compounds present in a diverse range of

plants used in traditional medicine and is known to have

antitumoral properties (Reyes-Zurita et al. 2009). This

group of hydrocarbons contains six isoprene units, wherein

methyl groups have been moved or removed, or oxygen

atoms added.

Maslinic acid (2-a,3-b-dihydroxiolean-12-en-28-oicacid), a triterpenoid compound present in the fruit and

leaves of Olea europaea, was found to act as a growth factor

when added to a standard trout diet (Fern�andez-Navarro

et al. 2008). Rainbow trout, Oncorhynchus mykiss, fed

diets containing maslinic acid at levels of 1, 5, 25 and

250 mg kg�1 for 225 days showed a higher white-muscle

weight and a protein-accumulation rate compared with fish

fed the control diet without maslinic acid supplementation.

Additionally, the total content of DNA, RNA and protein

in trout fed with 25 and 250 mg of maslinic acid per kg diet

were significantly (P < 0.05) higher than in the control,

and fractional and absolute protein synthesis rates

increased to more than 80% over the control values in these

two groups. Based on their results, the authors suggested

that maslinic acid may exert a strong anabolic effect on the

protein metabolism of fish and may even be useful as a feed

additive to stimulate white muscle growth in other organ-

isms. The results of the study have been reported to

complement their previous observation in rainbow trout

liver (Fern�andez-Navarro et al. 2006) and the authors

speculated that maslinic acid could stimulate the formation

of new cells by stimulating biosynthetic pathways of DNA,

RNA and protein, similar to those produced by a growth

factor.

Phytoandrogens

Some substances produced in plants may have functional

effects similar to testosterone in animals and are collectively

known as phytoandrogens (Turan & Akyurt 2005a).

Diosgenin [(25R)-5-spirosten-3b-ol], a steroid sapogenin

constituent of fenugreek seeds; daidzein, an isoflavone

present in soy; and triterpenoids isolated from the gutta

percha tree have been demonstrated to act as phytoandro-

gens (Raju et al. 2004; Chen & Chang 2007; Ong & Tan

2007). Phytoandrogens have been implicated in sex-reversal

in fish (Godwin et al. 2003).

Significantly higher (P < 0.05) weight gain, better FCR

and PER, and protein and lipid contents were observed in

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Phytochemicals in fish culture

C. gariepinus fed on feeds containing a phytoandrogen,

androstenedione (occurring naturally in pollens from

certain pine trees and European cactuses) at a concentra-

tion of 50 mg kg�1 diet for 120 days (Turan & Akyurt

2005a). The apparent net protein utilization (ANPU) was

also better in the phytoandrogen-fed fish. Phytoandrogens

may increase muscle mass, strengthen bone development

and such anabolic effects may be associated with fish

growth. Phytoandrogens have been applied safely in

humans. The therapeutic application of phytoandrogens

has been found rapidly to potentiate aspects of androgen

receptor (AR)-mediated anabolism, and unlike anabolic

steroids, phytoandrogenic modulators of the AR have

been reported not to aggravate arterial tension (Ong &

Tan 2007; Ong et al. 2011). However, like any other

phytoconstituents, the safe treatment regime for the

application of phytoandrogens in finfish culture must be

carefully analysed depending on the source plant and the

concerned fish species.

Plants with multibioactive compounds

Recently, a growing interest has emerged in using plants

containing numerous bioactive phytoconstituents in finfish

feed to promote growth. Dietary supplementation of differ-

ent parts of the plants and its extract based on sources such

as aqueous, methanol, ethanol, acetone and hexane has

been applied to induce growth promotion in fish. But the

isolation and characterization of bioactive principles must

be carried out in order to comprehend the functional

activity of the plants.

Tilapia, Oreochromis mossambicus, fed diets supple-

mented with acetone extracts (1% w/w) from medicinal

plants Cynodon dactylon, Aegle marmelos, Withania somnif-

era and Zingiber officinale for 45 days showed a significant

increase (P < 0.05) in mass and SGR compared with those

that received the control diet without any herbal supple-

ments (Immanuel et al. 2009). Moreover, the plasma pro-

tein, albumin, globulin, cholesterol, glucose and

triglyceride levels of the experimental fish were significantly

higher (P < 0.05) than that of the control fish. These herbs

contain different alkaloids, coumarins, triterpenoids,

b-sitosterol, steroidal lactones and volatile oils as potent

bioactive substances that may influence digestive processes

by enhancing enzyme activity, improving digestibility of

nutrients and feed absorption, thereby resulting in an

increase of fish growth (Samy et al. 2008; Immanuel et al.

2009; Citarasu 2010; Kaleeswaran et al. 2010; Hashemi &

Davoodi 2011).

The oriental medicinal herb, Glycyrrhiza glabra (liquo-

rice) comprising flavonoids and pentacyclic triterpene

saponins including liquiritin, liquiritigenin, isoliquiritigenin,

liquiritin apioside, glycyrrhizin and glycyrrhizic acid as

major constituents, was reported to have a growth-promot-

ing effect in Indian major carp, Cirrhinus mrigala, finger-

lings (Kumar et al. 2007). The fish were fed on feeds

containing root powder of the herb at either 0.1%, 0.2% or

0.3% levels for 60 days and the fish fed at the 0.3% herbal

inclusion level showed the highest (+24% of initial weight)

weight gain. Moreover, all the herbal diet fed fish showed

significantly (P < 0.05) better growth per day in% body

weight, average weight gain, FCR, gross conversion effi-

ciency compared with the control diet fed fish. Being rich

in isoflavones, the herb is shown to possess a plethora of

biological activities including potent antioxidant activity,

inhibition of superoxide anion production in xanthine/

xanthine oxidase system (Fu et al. 2004), protection of

mitochondrial function against oxidative stresses (Haragu-

chi et al. 2000) and thus may have contributed to general

health promotion in the fish.

By means of activity-guided fractionation of phytochem-

icals, Lee et al. (2005) demonstrated the supplemental

effects of maca meal (powdered tuber of Lepidium meyenii)

and its components extracted by four different solvents

(hexane, dichloromethane, ethyl acetate and methanol) on

the growth performance of juvenile rainbow trout. Fish

were fed on casein-based semipurified diets supplemented

with 15% maca meal or different extracts added to be

equivalent to that contained in a 15% portion of maca meal

for 14 weeks and improved growth was observed in fish fed

diets supplemented with maca meal or its methanol extract.

Different phytochemicals such as campesterol, stigmasterol,

b-sitosterol, quercetin, benzyl isothiocyanates, catechins

and other glucosinolates present in maca meal have been

reported to have many biological activities in fertilization,

immunostimulation, anabolism and balancing hormones;

and may have stimulated growth hormone in the fish lead-

ing to increased growth. The authors opined that being

highly polar, these compounds of interest could be

extracted by methanol. Moreover, the authors have attrib-

uted the higher growth of the fish fed maca meal supple-

mented diet to the attractants present in the maca meal,

which resulted in a significantly (P < 0.05) increased feed

intake. In previous studies also, the authors have demon-

strated that maca meal supplementation improved the pal-

atability of a semipurified diet and thereby increased the

intake, growth and feed utilization in rainbow trout juve-

niles at the first stage of exogenous feeding (Dabrowski

et al. 2003; Lee et al. 2004).

The growth inducing effect of a medicinal herbs mixture,

Massa medicata, Crataegi fructus, Artemisia capillaries and

Cnidium officinale (in 2:2:1:1 proportions, respectively)

containing numerous antioxidant compounds such as

carotinoids, flavonoides, cinnamic acids, benzoic acids,

folic acid, ascorbic acid, tocopherols and tocotorienols as

principle bioactive phytochemicals, was investigated in

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S. B. Chakraborty et al.

Japanese flounder, Paralichthys olivaceus (Ji et al. 2007a).

In the 8-week feeding trial, fish fed with 0.3%, 0.5% and

1.0% herbal mixture diets showed higher weight gain and

feed efficiency compared with fish in the control and 0.1%

herbal mixture fed groups. However, no significant

differences were found in survival, feed intake, final carcass

proximate composition, haemoglobin and haematocrit

levels, plasma total cholesterol level and alanine amino-

transferase activity among the dietary treatments.

Kelp grouper, Epinephelus bruneus, fed a diet supple-

mented with ethanol extract of the mushroom Phellinus

linteus for 30 days showed a significantly higher (P < 0.05)

percentage weight gain and feed efficiency compared with

fish fed the control diet without mushroom extract

(Harikrishnan et al. 2011b). The edible mushroom

contains a large number of biologically active compounds

such as polysaccharides and triterpenes that may exhibit

immunomodulating properties and act as a prebiotic.

Prebiotics are reported to be suitable for enhancing the

growth and activities of probiotics, bifidobacteria and

lactobacilli, and to suppress the growth of clostridia and

bacteroides, ultimately leading to stimulation of general

health and a growth increase in fish (Wang 2009; Ringo

et al. 2010).

Mechanisms of Action of Phytochemicals asGrowth Promoting Agents

Phytochemicals are in general considered ‘health promot-

ing’ by virtue of their antioxidant activity and positive

modulation, either directly or indirectly, of the cellular and

tissue redox balance (Liu 2003). A multitude of plants,

plant products and/or phytochemicals, in particular flavo-

noids, have been considered able to modulate cellular

responses to various stimuli interacting with reactive

oxygen-nitrogen species (RONS)-mediated intracellular

signalling either by scavenging reactive oxygen species and

suppressing their generation or by protecting antioxidant

defences and upregulating intracellular signalling resulting

in the antioxidant cellular response (Virgili & Marino

2008). This mechanism of twofold antioxidant activity may

also be assumed for health benefit in fish and leading to a

growth increase.

Medicinal herbs containing diverse groups of phyto-

chemicals such as phenolics, flavonoids, alkaloids, polysac-

charides, volatile oils and proteoglycans have been reported

to act as antimicrobial agents and to stimulate both specific

and non-specific immunity in fish by modulating the func-

tions of the immune cells, including T-cells, B-cells, NK-

cells and macrophages, increasing cytokine production and

immune related gene expression, and increasing antibody

production (Citarasu 2010; Chakraborty & Hancz 2011;

Pandey et al. 2012). Such immunostimulating properties of

herbs may lead to better health condition, disease resistance

and ultimately faster growth in fish.

However, the in vivo mechanism of action of phyto-

chemicals is recognized to be far more complex and plant

bioactive compounds with putative antioxidant capacity

have been demonstrated to perform activities and roles

independent of such capacity, interacting with cellular

functions at different levels. Phytochemicals may provide

health benefits as substrates for biochemical reactions,

cofactors as well as inhibitors of enzymatic reactions,

absorbants/sequestrants that bind to and eliminate undesir-

able constituents in the intestine, and compounds that

enhance the absorption and/or stability of essential nutri-

ents (Holst & Williamson 2008; Virgili & Marino 2008).

These effects of phychemicals have also been inferred to

promote growth in fish (Makkar et al. 2007; Immanuel

et al. 2009). The histophysiology of fish intestine plays an

important role in the digestive and absorptive functions of

the alimentary tract, thereby showing a significant effect on

fish nutrition and growth (El-Bakary & El-Gammal 2010).

Polyphenolic phytochemicals may also act either as elective

ligands or ligand mimics that agonize or antagonize cell

surface or intracellular receptors (Virgili & Marino 2008).

They also have been reported to promote DNA, RNA and

protein synthesis, stimulate GH and IGF-I production and

function and other anabolic effects in fish, resulting in

growth increase (Lee et al. 2005; Turan & Akyurt 2005a,b;

C�ek et al. 2007b; Fern�andez-Navarro et al. 2008; Goda

2008; Citarasu 2010).

Intestinal microflora provide several nutritional benefits

and play important roles in affecting the health of the host

fish (Burr et al. 2005; Yousefian & Amiri 2009; Zheng et al.

2009; Ringo et al. 2010). A wide variety of taxa such as

Acinetobacter, Enterobacter, Escherichia, Proteus, Serratia

etc. has been associated with the digestive tract of adult

freshwater fish (Austin 2006). These microorganisms have

been reported to possess the ability to produce enzymes

and to degrade complex molecules thereby exercising a

potential nutritional benefit, to produce vitamins and poly-

mers, to decompose esters of sulphuric acid thereby assist-

ing digestion and metabolism, and to utilize a variety of

resources such as citrate and sugars (Dhevendaran & Maya

2002; Austin 2006; Rudresh et al. 2010). The maintenance

of a healthy microbial ecosystem, intestinal epithelial integ-

rity, important host–microbe interactions at the mucosal

interface and the subsequent localized immunological

responses is paramount in protecting the host from enteric

infections, maintaining effective gut functionality and gen-

eral well being (Laparra & Sanz 2010). Phytochemicals and

their metabolic products may provide health benefits as

selective growth factors and fermentation substrates for

beneficial gastrointestinal bacteria, while acting as selective

inhibitors of deleterious intestinal bacteria, thereby exerting

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Phytochemicals in fish culture

prebiotic-like effects in fish (Zheng et al. 2009; Harikrish-

nan et al. 2011b). Thus, the cultured fish are conferred with

enhanced growth performance, feed efficiency and disease

resistance.

Interestingly, the effect of these dietary compounds is

influenced by the active bioavailable dose, rather than the

dose applied. Differences in the growth response to a phy-

tochemical may be due to differences in fish species’ ability

to metabolize or utilize a particular substance to which it

has been exposed in the diet (Pollack et al. 2003). A bio-

available dose may cause different magnitudes of effects in

different individuals and the maximum benefit may be

obtained at an optimal amount, while both deficient and

excessive levels may cause deleterious effects (Holst &

Williamson 2008). This may explain the variable effects of

phytochemicals on certain endpoints in different species

and differential levels of growth performance by a particu-

lar fish species at various concentrations of the phytochem-

ical. Moreover, the functional effects of different medicinal

herbs may often be associated with the extraction solvent

used and a synergistic effect of several phytoconstituents,

rather than the individual influence of any one major

bioactive principle (Lee et al. 2005; Harikrishnan et al.

2009; Zheng et al. 2009). However, further research works

are warranted to isolate and characterize the active

compounds from different plants, determine the ideal

concentration for dietary administration of phychemicals

as growth promoters and to analyse the detailed functional

mechanism behind the growth-promoting effects of plant

bioactive compounds in different finfish species in order to

establish the potential of these vast arrays of phytoconstitu-

ents for field evaluations in the practice.

Phytochemicals as Endocrine Modulating Agents

Sexual size dimorphism and dichromatism are characteris-

tics of many animal species including fish. Manipulation of

phenotypic sex in fish farming is generally desirable since

one gender, depending on the species, grows faster (Uguz

et al. 2003). In addition, the need for high quality fish seed

has necessitated research into various ways of enhancing

fertility to meet the growing demand. Ornamental fish

trade plays a significant role in the economy of many devel-

oped and developing countries. In many groups of orna-

mental fish, the sexes differ in their colour patterns, with

males being the more brightly coloured sex (Cogliati et al.

2010). Synthetic steroids are commonly used for such

manipulation of sex and as enhancers of fertility in fish, but

because of their potential hazards the use of phytochemi-

cals is a potential alternative to be explored. The use of

medicinal plants as fertility enhancers and sex reversal

agents in fish has been receiving some attention. Moreover,

as the components of fish diet and/or compounds present

in the aquatic environment, phytochemicals may induce

biological responses in fish including oestrogenic effects

and reproductive retardance, and hence are sometimes

regarded as endocrine disrupting chemicals (EDCs)

(Ng et al. 2006; Cheshenko et al. 2008). The presence of

the egg yolk glycoprotein vitellogenin (Vtg) in male or

juvenile fish has become a powerful and well established

biomarker for the evaluation of xenoestrogens (Liao et al.

2011). Hepatic ethoxyresorufin-O-deethylase (EROD)

activity related to the rate of the enzyme Cytochrome P450

subfamily 1A (CYP1A), which is critical for oestrogen

metabolism, is also a highly sensitive in vivo bioindicator of

exposure to xenobiotic chemicals (Green & Kelly 2009). In

this respect also, the use of phytochemicals in fish culture

needs to be investigated.

Saponins

Saponins have been found to modulate the sex ratio of fish

in few instances. Francis et al. (2002b) reported that dietary

Quillaja saponin had the potential to change the sex-ratio

in favour of males in Nile tilapia. Seventeen-day-old Nile

tilapia fry were fed diets containing different concentrations

of QS extract (50, 150, 300, 500 and 700 mg kg�1) for

6 months and the sex ratio of the fish in the QS fed groups

deviated from the normal 1:1 ratio, with the group fed QS

supplemented diet at a concentration of 700 mg kg�1

showing a significantly higher number of males (number of

males: number of females = 22:10, P < 0.05). The authors

demonstrated that QS promoted luteinizing hormone (LH)

release from cultured pituitary cells of tilapia, but in vivo

the average LH values did not show any trends with respect

to the effect of dietary QS. Based on the results obtained,

the authors concluded that dietary QS might have exerted

its effect on the sex ratio of tilapia via interference in either

the follicle stimulating hormone (FSH) at the pituitary level

or the androgens at the gonadal level.

Different concentrations (0.1, 0.2 and 0.3 g L�1) of

T. terrestris extract (containing steroidal saponins as princi-

ple phytoconstituents) were tested for their effect on sex

reversal in convict cichlid by immersing newly hatched off-

spring once weekly for 2 months in T. terrestris extract

(C�ek et al. 2007a). Sex ratios in all the treatment dosages

were significantly (P < 0.001) different from the expected

1:1 ratio, while treatment with T. terrestris extract at a con-

centration of 0.3 g L�1 was the most effective in terms of

masculinization, producing a maximum (~87%) percent-

age of males.

A dose dependent increase in the percentage of males

was observed in C. gariepinus at the end of a 30 day

immersion treatment (thrice weekly) with 3, 6 and 9 g

30 L�1 T. terrestris extracts (Turan & C�ek 2007). But, the

highest concentration of the extract applied in the study

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S. B. Chakraborty et al.

yielded ~80% males and it was speculated that application

of a higher concentration might have resulted in a higher

percentage of males in the fish population.

Similarly, immersion of newly born guppy in T. terrestris

extract at a concentration of 0.15 g L�1 once weekly for

2 months resulted in 80% (P < 0.01) male fish (C�ek et al.

2007b). Although, statistical analysis revealed no significant

difference (P > 0.05) in the sex ratios of two lower concen-

trations (0.05 and 0.1 g L�1) of T. terrestris extract from

the expected 1:1 ratio, treatment with both these concentra-

tions resulted in a higher percentage of males (58.25% and

59.77% for 0.05 and 0.1 g L�1, respectively) compared with

the control. Histological examinations demonstrated that

treatment with T. terrestris extract stimulated spermatogen-

esis in both fish species. However, the inability of the plant

extract even at the highest applied concentration, for pro-

ducing 100% male population of P. reticulata opens scope

for further studies in lieu of determining the ideal treat-

ment regime with this extract. Protodioscin, the most dom-

inant saponin in T. terrestris, has been reported to enhance

testosterone production and to elevate the levels of dehy-

droepiandrosterone, dihydrotestosterone and dehydroepi-

androsterone sulphate in animals (Gauthaman & Ganesan

2008), and this increased concentration of androgens may

be considered responsible for the masculinizing effect of

the extract in fish.

A dose dependent masculinization effect of T. terrestris

extract has been reported in another freshwater ornamental

fish Poecilia latipinna also (Kavitha & Subramanian 2011;

Kavitha et al. 2012). New born hatchlings of the fish were

immersed in different concentrations of the T. terrestris

extract (100, 150, 200, 250 and 300 mg L�1) for 2 months

and an increase in total body weight, testis weight and sper-

matogenesis was observed. Moreover, the activities of

testicular functional enzyme alkaline phosphatase (ALP),

acid phosphatase (ACP), sorbitol dehydrogenase (SDH),

lactate dehydrogenase (LDH) and glucose-6-phosphate

dehydrogenase (G6PDH) levels were found to change to a

different extent in treated groups compared with that of

the control. The authors suggested that T. terrestris might

have induced testicular enzyme activity that might aid in

male reproductive functions. The extract of T. terrestris has

been used as masculinizing agent and its application has

also resulted in better growth of the treated fish. But the

optimum concentration of the extract has been found

variable for induction of the maximum growth increase

and sex reversal. Thus, during its application, the concen-

tration of the extract should be determined according to

the major objective of the study. In addition, several studies

have indicated the masculinizing effect of T. terrestris

extract in fish during immersion experiments but extensive

research is required for its dietary administration during

commercial fish culture.

Phytosterols

Phytosterols are a group of steroid alcohols with chemical

structures similar to cholesterol obtained in plants. The role

of phytosterols in the modulation of endocrinal homeosta-

sis of fish has evoked great attention. b-Sitosterol is one ofthe most common phytosterols present in aquatic environ-

ments receiving pulp and paper mill effluents (Orrego et al.

2010). It is also one ingredient in soy bean extracts used for

many commercial fish diets (Nakari & Erkomaa 2003).

Other phytosterols include campestrol, stigmasterol and

stigmastanol (Gilman et al. 2003). Several studies have

been conducted to determine the importance of this plant

bioactive principle as endocrine disrupting chemicals.

b-Sitosterol was reported significantly (P < 0.05) to

elevate plasma vitellogenin levels, reduce plasma cholesterol

and pregnenolone levels, but show little or no effect on

testosterone levels in sexually immature rainbow trout

(Tremblay & Vanderkraak 1999). Multiple intraperitoneal

injections with b-sitosterol (one injection every 7 days for a

total exposure period of 28 days) showed significant induc-

tions of ethoxyresorufin-O-deethylase and increased

vitellogenin levels in immature rainbow trout (Orrego et al.

2010).

Male brook trout, Salvelinus fontinalis, exposed for

21 days to the b-sitosterol preparation at a concentration

of 100 lg b-sitosterol preparation g�1 of fish via slow-

release Silastic intraperitoneal implants had lower plasma

levels of sex steroids and cholesterol, and lower in vitro

gonadal sex steroid production; but the activity of the

enzyme P450 side-chain cleavage (P450scc) was not

affected in testis mitochondria isolated from the fish

(Gilman et al. 2003).

Similar results were observed in goldfish, Carassius aura-

tus, exposed for 31 days to the b-sitosterol preparation at

concentration of 150 lg b-sitosterol preparation g�1 of

fish during the same experiment (Gilman et al. 2003).

Interestingly, while investigating the effects of b-sitosterolon the reproductive fitness of goldfish, Maclatchy and Van-

derkraak (1995) found significantly (P < 0.05) decreased

plasma testosterone levels in both male and female fish,

decreased 17 b-oestradiol levels in female, decreased 11-

ketotestosterone levels in male fish, but elevated plasma

gonadotropin (GtH)-II levels in males at day 4 following

two intraperitoneal injections with b-sitosterol.Phytosterols containing b-sitosterol were shown to dis-

rupt the reproduction system of zebrafish, Danio rerio

(Nakari & Erkomaa 2003). A mixed sex population of the

fish was exposed continuously across three generations to

wood sterol (containing 80% b-sitosterol) at concentra-

tions of 10 and 20 lg L�1, and soy sterol (containing ~50%b-sitosterol) at a concentration of 10 lg L�1. Both sterol

preparations caused vitellogenin induction in the exposed

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Phytochemicals in fish culture

fish, while wood sterol changed the sex ratio of the exposed

fish. In generation F1, the predominant sex was male, and

in generation F2 it was female. The two phase response

indicates the possibility that hormonal dysfunctions

induced by phytosterols are transmittable by DNA. The

results indicate multiple possible mechanisms for phytos-

terol induced inhibition of gonadal steroidogenesis in fish.

Flavonoids

Another group of important endocrine disrupting chemi-

cals is flavonoids. Genistein, a well-characterized isoflav-

one, is an important secondary plant metabolite that has

been reported to be present in soybean meal and pulp mill

effluents (Green & Kelly 2009), and there are numerous

reports of genistein exerting oestrogenic effects in fish.

Moreover, the functional group of phytoestrogens is

reported to have a variety of hormonal and gametic effects

in a range of fish species.

Evaluating the effects of genistein enriched diets on the

endocrine process of gametogenesis and reproduction effi-

ciency of rainbow trout, Bennetau-Pelissero et al. (2001)

reported increased plasma vitellogenin concentrations in

male and female fish. Fish undergoing their first gametogen-

esis were fed diets containing either 500 or 1000 ppm geni-

stein until spawning. In males, a slight but constant

induction of vitellogenin synthesis and a decrease in testos-

terone levels were observed, while in females, a significant

(P < 0.05) increase in plasma vitellogenin occurred only at

the beginning and at the end of oogenesis. Male fish fed a diet

with 500 ppm genistein showed a slight decrease in plasma

levels of bFSH and bLH at the end of spermatogenesis, testic-

ular development was accelerated in genistein-fed fish, and

sperm motility and concentration were decreased in a dose-

dependent manner at spawning. Females fed genistein

supplemented diets showed decreased bFSH and bLH levels,

whereas delayed spawning and impaired gamete quality

underlined by lower percentage of ovulating females, lower

fertilization rate and lower viability of fry were observed in

females fed the diet containing 500 ppm genistein.

Genistein was fed to striped bass, Morone saxatilis, fin-

gerlings at various concentrations (2.0, 4.0 and 8.0 mg g�1

of diet) and significantly higher (P < 0.05) levels of vitel-

logenin were observed in 2.0 and 8.0 mg g�1 doses com-

pared with the 4.0 mg g�1 and control group, while the

4.0 mg g�1 dose showed no significant difference from

the control (Pollack et al. 2003). The results indicated that

the juvenile striped bass responded to the oestrogen-like

function of genistein in a manner characteristic of the low-

dose effects of some endocrine disrupting chemicals.

But in yellow perch, Perca flavescens, Ko et al. (1999)

reported no apparent oestrogenic effects of genistein (0.75

and 7.5 mg g�1 diet) on reproductive function.

Interestingly, increased proportions of male and intersex

individuals were observed in Channel catfish fed diets con-

taining genistein at 4 and 8 mg g�1 concentration between

5 and 140 days posthatch, while there was no significant

(P > 0.05) difference in EROD activity between the control

and treated groups (Green & Kelly 2009). Phenotypic sex

was found to be significantly dependent on the dietary phy-

toestrogen concentration (P = 0.01) and a significant rela-

tion existed between genistein concentration in the diet

and gonadal sex (P = 0.02). This paradoxical sex reversal

might have resulted from the dual role of genistein as not

only an oestrogen agonist but also as an antagonist block-

ing oestrogen’s action.

Immersion of newly hatched C. gariepinus larvae in a

commercial mixture of phytoestrogens at a concentration

of 1500 mg mixture 30 L�1 water every 3 days for 30 days

produced ~70% females, while lower concentrations (210,

420, 630 and 750 mg mixture 30 L�1 water) had no effect

on the sex ratio (Yılmaz et al. 2009). The authors suggested

that the usage of higher doses and treatment durations of

the mixture could be more effective for all-female produc-

tion of the African sharptooth catfish population. However,

the authors did not measure plasma oestrogen levels during

the experiment, and hence could not deduce whether this

potency was caused due to an increase in oestrogen level in

the fish.

Inudo et al. (2004) assessed the effects of differing die-

tary phytoestrogen content (including genistein and daidz-

ein) in Japanese fish diets on the hepatic vitellogenin

production and reproduction in medaka, Oryzias latipes. At

the end of the 28 day feeding trial, there was no significant

difference in fecundity and fertility between the fish fed

diets containing high and low levels of phytoestrogens.

However, hepatic vitellogenin values were significantly

(P < 0.05) higher for male medaka fed a diet containing

high phytoestrogen concentration (genistein, 58.5 lg g�1;

daidzein, 37.3 lg g�1) compared with that in fish fed diets

with a low concentration of phytoestrogens (genistein, <0.8lg g�1; daidzein, <0.8 lg g�1 and genistein, 1.4 lg g�1;

daidzein, 2.0 lg g�1). The findings indicate that high

amounts of phytoestrogens have the potential to induce

hepatic vitellogenin production in male medaka.

On the other hand, neither a sex reversal nor the induc-

tion of vitellogenin expression could be detected in just-

hatched medaka exposed to the isoflavonoid naringenin

and its derivatives at concentrations of 67 nM and 670 nM

for 23 days (Zierau et al. 2005).

The plasma vitellogenin concentration in goldfish fed

commercial diet containing a high amount of phytoestro-

gens, including genistein and daidzein, was about 100-fold

higher compared with that in fish fed diet with a low

phytoestrogen concentration (Ishibashi et al. 2004). The

authors opined that phytoestrogens, at high concentrations,

Reviews in Aquaculture (2013) 5, 1–19

© 2013 Wiley Publishing Asia Pty Ltd12

S. B. Chakraborty et al.

might inhibit the synthesis and/or release of testosterone

and 11-ketotestosterone.

Phytochemicals such as daidzein, biochanin A, genistein,

equol and coumestrol showed estrogenic activity as assessed

by their induction of hepatic Vtg synthesis during intraperi-

toneal administration to yearling Siberian sturgeon, Acipenser

baeri (Bennetau-Pelissero et al. 1991a). Coumestrol seemed

to be themost potent compound, inducing the highest level of

vitellogenin secretion with the lowest dose administered.

Soybean-based diets containing different isoflavones were also

reported to increase plasma vitellogenin concentrations in

Siberian sturgeon (Bennetau-Pelissero et al. 1991b).

Liao et al. (2011) compared vitellogenic responses in

Chinese rare minnow, Gobiocypris rarus, fed with different

diets. In juvenile stages, vitellogenin in the fish fed with

nauplii of Artemia sp. was significantly (P < 0.05) lower

than that in the fish fed with commercial pellet feed and

Tubifex sp. from a wastewater treatment plant. The EROD

activities in whole body homogenates of juvenile fish fed

with pellet feed and Tubifex sp. from a wastewater treat-

ment plant were significantly higher (P < 0.05) than that of

juvenile fish fed nauplii of Artemia sp. But in adult stages,

significant (P < 0.05) induction of vitellogenin was only

found in the males fed with pellet feed compared with

males fed with nauplii of Artemia sp. There was no signifi-

cant (P > 0.05) difference in vitellogenin induction in

females fed with different diets. There was no significant

difference (P > 0.05) in sex ratios and average fecundity

between the groups, but hatchability, fertilization and the

frequency of spawning were significantly lower in fish fed

with pellet feed compared with that in fish fed nauplii of

Artemia sp. Many oestrogenic pollutants have been found

in the pellet feed and Tubifex sp. from a wastewater plant

that may have caused vitellogenin induction and high

EROD activity, and dietary exposure of females to

endocrine disrupters may have impaired the reproductive

success of rare minnow.

Exposure of sexually mature male fighting fish,

Betta splendens, to pharmacological concentrations

(1000 lg L�1) of genistein and environmentally relevant

(1 lg L�1) concentrations of genistein and b-sitosterolsingly and in combination showed no significant effect on

circulating levels of androgen 11-ketotestosterone, oestro-

gen E2, gonadosomatic index, sperm concentration and

motility, or fertilization success (Stevenson et al. 2011).

Plants with multibioactive compounds

Herbs containing multiple bioactive principles including

flavonoids and sterols may act as potent EDCs and alter

fertility in fish.

Dietary supplementation (1%) of lowbush blueberry,

Vaccinium angustifolium, product that contains high

phenolic content including the flavonoids anthocyanins,

was found to inhibit lipid peroxidation in Arctic char,

Salvelinus alpinus, semen by decreasing the rate of sperm

lipid peroxidation and increasing the antioxidant potential

of seminal plasma (Mansour et al. 2006).

Adeparusi et al. (2010) investigated the effects of Kigelia

africana fruit on sperm quality of C. gariepinus. Fish were

fed diets containing K. africana root powder at concentra-

tions of either 50, 100, 150 or 200 g kg�1 diet for a period

of 90 days and milt qualities were assessed by microscopic

studies and fertility tests. The male brooder fish fed

K. africana root powder supplemented diet at a concentra-

tion of 100 g kg�1 diet had significantly (P < 0.05) higher

sperm counts, percentage motility and fertilization ability,

but lower milt volume and motility duration than the fish

fed the control diet without the medicinal herb. However,

there was no significant difference (P > 0.05) in the length

and weight of the testes among the diet groups. Consider-

ing the results, the authors concluded that K. africana fruits

might have promising pro-fertility in fish seed production.

Dada and Ajilore (2009) used the ethanol extract of

another medicinal herb, Garcinia kola seeds to enhance

fertility in C. gariepinus. Fish were fed diets supplemented

with different concentrations (0.25, 0.5, 1.0 and 2.0 g kg�1

diet) of ethanol extract of G. kola seeds for 56 days and a

significant difference (P < 0.05) was observed in fecundity.

The diameter of the eggs increased with the dose of ethanol

extract in the fish diet, while histological analysis revealed a

dose related increase in the alteration and degenerative

changes such as cytoplasm shrinkage, rupture of the cell

membrane and different vacuole sizes. Based on the obser-

vations, dietary supplementation of ethanol extract of

G. kola seeds at the concentrations between 0.25 and

0.5 g kg�1 diet were recommended for fertility enhance-

ment in African catfish. The authors speculated that the

reason behind the increase in the fecundity of the herbal

supplemented diet fed fish was the presence of bioflavo-

noids and xanthone in the plant. The compounds are

potent antioxidants and can increase oestrogen production,

thereby leading to production and maturation of eggs.

Mechanism of action of phytochemicals as endocrine

modulating agents

Varied mechanisms including effects at the steroid receptor

level, effects on steroid synthesis, distribution and excre-

tion, actions on the hypothalamus–pituitary–gonad axis, as

well as indirect mechanisms including thyroid and growth

hormone disruption have been postulated for the repro-

ductive endocrine disruption in fish populations by phyto-

chemicals such as steroidal saponins, flavonoids and

phytosterols present in the environment and diet (Rempel

& Schlenk 2008). Saponins have been postulated to modify

Reviews in Aquaculture (2013) 5, 1–19

© 2013 Wiley Publishing Asia Pty Ltd 13

Phytochemicals in fish culture

the sex ratio in fish through interference in the pituitary–gonad axis and alteration of the secretion level of hypophy-

seal gonadotropins and gonadal androgens (Francis et al.

2002b; C�ek et al. 2007a,b). Phytosterols have been specu-

lated to induce reproductive dysfunction in fish by reduc-

ing the gonadal steroid biosynthetic capacity through either

effects on cholesterol availability or the activity of the

P450scc (Maclatchy & Vanderkraak 1995; Tremblay & Van-

derkraak 1999). Phytoestrogens including isoflavones such

as genistein and daidzein have been reported to bind

weakly to oestrogen receptors and either to produce or

inhibit oestrogen effects (Ziegler 2004; Green & Kelly

2009). Interestingly, in an in vitro assay, daidzein was dem-

onstrated to induce androgenic effects by modulating

androgen receptor coactivators (Chen & Chang 2007).

Moreover, numerous phytoestrogens have been reported to

be potent inhibitors of aromatase CYP19 by either acting as

competitive inhibitors with natural substrates for the

enzyme, decreasing cAMP-responsive element binding

protein (CREB) expression or inhibiting the generation of

cAMP, thus affecting one of the pathways for regulation of

aromatase expression and hence activity in fish (Cheshenko

et al. 2008). However, determination of the optimum

dietary level of different phytochemicals for maximum

effects and the complete understanding of a variety of

pathways associated with functional mechanisms of phyto-

compounds as endocrine disrupting chemicals causing

both masculinization and feminization warrant further

research work in these regards.

Conclusions

Despite the potential benefits to health and performance as

noted in various terrestrial species including human, scat-

tered information is available about the use of phytochemi-

cals in fish culture. Phytochemicals that can be used as

growth-promoters in fish species and for monosex fish

production may provide valid alternatives to synthetic

compounds. Essential oils, saponins, flavonoids and

phytosterols are found to be the most important classes of

phytochemicals that have drawn maximum attention in

this regard. The functional activities of these compounds

have been found to be dose dependent, exerting different

physiological effects at different concentrations. However,

gaps exist between the knowledge of the functional mecha-

nism behind the physiological activity, determination of an

ideal extraction solvent, treatment dose and duration

regime and mode of administration for different phyto-

chemicals to obtain the maximum benefit, and all these

aspects call for extensive future research. Moreover,

research on bioavailability and detailed metabolic processes

associated with the potential sex-reversing phytochemicals

must be expanded. The molecular mechanisms behind

EDC-induced disruption of biological processes, leading to

malfunctioning of the reproductive system in fish require

detailed characterization. Though biodegradable, consider-

ing the modulating effects of phytochemicals on the

hormonal system of fish, a cautious approach should be

taken before recommending their commercial application

during large-scale finfish culture and the need for legal

approval of such application may be debated. A compre-

hensive knowledge base regarding the residual effect, if any,

of these phytochemicals must be developed to assure the

safe human consumption of phytochemical-treated fish. In

conclusion, multifaceted and coordinated research efforts

need to be oriented in every respect to further increase the

use of phytochemicals in fish culture. Such efforts will

result in increasing the sustainability of fish culture.

Acknowledgements

Suman Bhusan Chakraborty holds the Scholarship for Post-

doctoral Study (Type D) in Hungary (M€OB/160-2/2010)

by the Hungarian Scholarship Board.

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