Effects of long term feeding of Quillaja saponins on sex ratio, muscle and serum cholesterol and LH levels in Nile tilapia (Oreochromis niloticus (L.))

Comparative Biochemistry and Physiology Part C 133(2002) 593–603

1532-0456/02/$ - see front matter� 2002 Elsevier Science Inc. All rights reserved.PII: S1532-0456Ž02.00167-9

Effects of long term feeding ofQuillaja saponins on sex ratio,muscle and serum cholesterol and LH levels in Nile tilapia

(Oreochromis niloticus (L.))

George Francis , Berta Levavi-Sivan , Ayelet Avitan , Klaus Becker *a b b a,

Department of Animal Nutrition and Aquaculture, Institute for Animal Production in the Tropics and Subtropics,a

University of Hohenheim (480), D 70593 Stuttgart, GermanyDepartment of Animal Sciences, Faculty of Agricultural, Food and Environment Quality Sciences, The Hebrew University,b

P.O. Box 12, Rehovot 76100, Israel

Received 2 May 2002; received in revised form 13 August 2002; accepted 17 August 2002

Abstract

Seventeen-day-old Nile tilapia fry were fed a standard diet(C) or diets containing 50–700 mg kg Quillaja saponiny1

(QS) extract(groups S50, S150, S300, S500 and S700). After the first 8 weeks, 30 randomly selected tilapia from eachof the treatments were placed in separate aquaria and fed the standard diet without saponins from then on(these weredesignated S50yC, S150yC, S300yC, S500yC and S700yC). The fish grew from an initial average weight of approximately30 mg to a final average weight of 79 g during the 6-month feeding period. The difference between the average weightof C-fed tilapia and the treatment with the highest average weight after 6 months was 53.5%. The sex ratio of tilapia inthe saponin-fed groups deviated from the normal 50:50 male:female ratio, with the S700 group showing a significantlyhigher number of males.Quillaja saponin stimulated LH release from dispersed tilapia pituitary cells in vitro. This effectwas abolished in the presence of dilute calf serum. Serum LH values did not show any diet-dependent trend in eithermale or female tilapia in vivo. In both continuously saponin-fed and only-initially saponin-fed groups, the average serum(but not muscle) cholesterol levels in males showed an increasing trend(R values of 0.62 and 0.69) with increasing2

dietary saponin level. It was concluded that dietary QS has the potential to change the sex-ratio in favour of males.More investigations are required to determine the mechanism of action and the optimum dietary level of QS formaximum effects.� 2002 Elsevier Science Inc. All rights reserved.

Keywords: Nile tilapia; Quillaja saponins; Sex-ratio; Growth; Leutinizing hormone; Cholesterol

1. Introduction

The saponins are naturally occurring surface-active glycosides mainly produced by plants, butalso by lower marine animals and some bacteria(Yoshiki et al. 1998; Riguera, 1997). Their abilityto form stable, soap-like foams in aqueous solu-

*Corresponding author. Tel.:q49-711-4593158; fax:q49-711-4593702.

E-mail address: [email protected](K. Becker).

tions attracted human interest from ancient times.Saponins consist of a sugar moiety usually con-taining glucose, galactose, glucuronic acid, xylose,rhamnose or methylpentose, glycosidically linkedto a hydrophobic aglycone(sapogenin), whichmay be triterpenoid or steroid in nature. Oneexample of an extensively studied group of triter-penoid saponins is produced fromQuillaja sapon-aria, a tree native to the Andes region. Water

594 G. Francis et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 593–603

extracts of the bark of this tree has been tradition-ally used by shamans as an overall curing agent.When present in the diet of animals, saponins

are believed to have several negative effects. Forexample, dietary saponins derived from differentplants have been held responsible for depressionof feed intake, reduction in weight gain, accentu-ation of ruminant bloat, photosensitization(Chee-ke, 1996), inhibition of the active uptake ofnutrients(Johnson et al., 1986) including vitamins(Jenkins and Atwal, 1994) and minerals(Southonet al., 1988) in the intestine, lowering of proteindigestibility (Shimoyamada et al., 1998), and forinducing infertility (Tewary et al., 1973; Quin andXu, 1998). On the other hand, saponins have alsolong been known to possess properties useful toman as they are the active components in a largenumber of traditional ‘herbal medicine’ prepara-tions. There have been reports of antiviral activityof saponins fromGlycyrrhiza radix, cholesterollowering activity of saponins from soybean, immu-nostimulant activity of saponins fromQuillajasaponaria Molina, and hypoglycaemic and anti-diabetic activity of saponins from fenugreek(Ken-sil, 1996; Petit et al., 1993). Partially purifiedQuillaja saponaria Molina saponin preparationssuch as Quil A, have found wide-spread use inveterinary vaccines(Kensil, 1996). Saponins areimportant in human nutrition as well because oftheir widespread occurrence in food constituentssuch as legumes.Tilapia aquaculture is and will continue to be

important, particularly for the lesser-developedcountries in the tropics(FAO, 2001). A majordrawback of tilapia as a culture fish is their prolificreproduction, resulting in a harvested fish size,which is unacceptable in many markets. Treatmentwith steroid hormones has been applied to produceall-male populations, in order to circumvent thisproblem. Because of environmental and consumerhealth concerns(over 99% of hormones adminis-tered through the diet are released into the waterin less than 24 h; see review by Pandian andSheela, 1995), this treatment is prohibited in manycountries.Tilapia are also otherwise valuable as an impor-

tant experimental vertebrate model(Anken et al.,1993) and as a potential source of pancreatic isletsfor possible transplantation into diabetics(Mac-Kenzie, 1996). Studies on the physiology of thesefish are, therefore, of high scientific interest.

In previous experiments, we observed that die-tary Quillaja saponins(QS) stimulate growth incarp (Francis et al., 2002) and in addition inhibitegg production by female Nile tilapia(Francis etal., 2001 and unpublished). The objectives of thecurrent experiment were to study the effects of QSin the diet on sex ratio in Nile tilapia fry, andwhen tested on LH secretion from tilapia pituitarycells in vitro. Observations were also made on thelevel of muscle and serum cholesterol levels ofthe fish.

2. Materials and methods

2.1. Experimental feeds

There were six experimental diets designated C(control), S50, S150, S300, S500 and S700 pre-pared from the same basal diet to ensure uniformcomposition. The basal diet contained approxi-mately 40% protein, 10% lipid, 10% ash and had20 kJ g gross energy on a dry matter basis. They1

ingredients were as previously reported(Francis etal., 2001). The basal diet was pelleted(to approx.2 mm diameter) with no additions in the case ofthe control feed(C). Saponin(QS; Sigma no. S-2149, Sigma, St. Louis, MO, USA), dissolved indemineralized water, was mixed thoroughly(at 50,150, 300, 500 and 700 mg kg basal diet in they1

S50, S150, S300, S500 and S700 diets, respective-ly) with the powdered feed using a mixer beforepelleting. The moist pellets were frozen, freeze-dried and stored in a freezer(y18 8C) until use.For the in vitro LH production experiments fromdispersed tilapia cells, QS, and its purified form(QS-P, Sigma no. S-4521) was used.

2.2. Experimental fish and feeding regime

Approximately 600 14-day-old tilapia fry wereobtained from the ‘Institut fur Tierzucht und Haus-tiergenetik’, Georg-August Universitat, Gottingen,¨ ¨Germany. They were fed a commercial(‘Tetramini’) flaked feed for 3 days to allow them torecover and acclimatise to conditions in the lab-oratory. They were then divided into seven treat-ment groups of 70 fish each. Two of the sevengroups were fed control(C) feed and each of thefive remaining tanks were provided the S50, S150,S300, S500 and S700 diets, respectively. The fishwere fed at the rate of 20% of body mass till theyreached 0.8 g, then 12% of body mass till they

595G. Francis et al. / Comparative Biochemistry and Physiology Part C 133 (2002) 593–603

weighed on average 3.5 g each, followed by 6%of body mass till the average weight was approx-imately 12 g. Afterwards, they were fed at a levelof 20 g per kg metabolic body mass(kg ) and0.8

was further reduced, when they reached approxi-mately 25 g body mass, to 10 g per kg . After 80.8

weeks of feeding the experimental diets, 30 tilapiafrom each of the five treatment groups were placedin separate tanks and fed the C diet. These groupswere designated S50yC, S150yC, S300yC, S500yC and S700yC, respectively, based on the diet oftheir original group. Simultaneously, the two con-trol groups were split each into two separate groupsto equalize tank-density.During the experiment, the fish in each treat-

ment were kept in 75-l aquaria that were part of amain recirculating system, under a 12 h-lighty12-h darkness regime. The water in the aquaria withsaponin-containing feed was allowed to flowthrough to avoid possible contamination in therecirculating system. The fish were weighed onceevery two weeks and the feed ration adjustedaccording to body mass. Water flow was adjustedto keep oxygen saturation constantly above the75% level. The water temperature in the experi-mental system was maintained at 27"0.1 8C. Allwater-quality parameters were maintained atacceptable levels(pH: 7–7.9; total NH3: 0.1–0.2mg l ; nitrite: 0.07–0.1 mg l and nitrate: 1–3y1 y1

mg l ) throughout the experiment.y1

2.3. Measurements and analyses

Three male and three female fish from eachgroup were taken out after 5 months to draw bloodfor hormone analysis. Blood was drawn from thehaemal arch of each fish using disposable syringesand was stored on ice until the serum was sepa-rated by centrifugation(10 000=g for 5 min). Theserum was stored aty20 8C until LH radioim-munoassay(RIA). These fish were then discarded.After approximately 6 months(June 2000 to

November 2000) of feeding the experimental diets,the fish in each treatment group were first collec-tively weighed and the number of males andfemales in each group counted. Sexing was basedon the shape of the genital papilla, which is broadin females and pointed in males. The male fishalso produced milt upon gentle squeezing of theabdomen. The Specific Growth Rate(SGR) wascalculated using the formula{ wLN(W )-LN(Wt)xy0

t} *100, where LN(W ) and LN(Wt) are natural0

logarithms of initial and final weights andt is thetime interval in days.Five males and five females were again random-

ly selected from each group. The sample numberwas kept low in order to have enough fish forsubsequent breeding experiments. Blood wasdrawn from the sampled fish as previouslydescribed. The serum was stored aty20 8C untilRIA and cholesterol assay. Serum cholesterol wasmeasured using a cholesterol estimation kit(RocheDiagnostic, Mannheim, Germany). Five grams ofmuscle tissue were then taken from each fish andstored aty18 8C for determining muscle choles-terol content(using a cholesterol estimation kit;Boehringer Mannheim, Germany).

2.4. In-vitro stimulation of LH secretion

Pituitary cells were dispersed according to Leva-vi-Sivan and Yaron(1992) and Levavi-Sivan etal. (1995). Briefly, pituitary cells from 80 to100fish were collectively dispersed, counted and thenplated at 100 000 cells per well of 96-well plate,in 0.2-ml medium (M199,10% fetal calf serum(FCS), 10 mM HEPES, 1% antibioticswPen-strep-nystatin suspensionx; Biological Industries, BeitHa’emek, Israel), and cultured for 4 days at 288Cunder 5% CO . On the fourth day, the cells were2

rinsed twice with medium before addition of thetest substances in stimulation medium(M199, 1%antibiotics, 0.1% BSA), for 5 h. Salmon GnRH(Trp ,Leu -LHRH; sGnRH) and QS were dis-7 8

solved directly in the medium and were preparedfresh. The concentration of sGnRH used was 100nM, the most effective dose in raising taGtH levelsin dispersed tilapia pituitary cells(Levavi-Sivan etal., 1995). 20% FCS was added to the stimulationmedium instead of the BSA to test the activity ofQS in the presence of serum.

2.5. Radioimmunoassay

The gonadotropin of tilapia(taGtH), possiblyrepresenting the tilapia LH was determined in themedium by specific RIA according to Levavi-Sivan and Yaron(1992). The second antibodyemployed was donkey anti-rabbit IgG bound tomagnetizable compound(Amerlex-M, Amersham,UK). The sensitivity of the assay was 0.5 ngytube; the intra- and interassay coefficients of var-iation were 7.3% and 14%, respectively. The taGtHwas measured by homologous RIA, according to


Table 1Total number(N), average body weight and SGR of tilapia inthe various treatments after 6 months

Treatment N Average body SGRweight (g) (% per day)

C1 25 64.3 3.00C2 24 68.2 3.03C3 22 54.6 2.91C4 23 65.2 3.01S50yC 23 73.1 3.07S150yC 20 91.2 3.19S300yC 24 86.1 3.16S500yC 23 64.4 3.00S700yC 19 75.2 3.09S50 33 78.9 3.11S150 25 98.1 3.23S300 28 96.4 3.22S500 29 98.0 3.23S700 26 94.3 3.21

C-tilapia fed control feed; S50yC to S700yC-tilapia fed dietscontaining 50, 150....up to 700 ppmQuillaja saponins for 8weeks(week 3 to week 11) followed by control; S50 to S700-tilapia fed diets with 50, 150...up to 700 ppmQuillaja saponinsthroughout.

Bogomolnaya et al.(1989), using taGtH purifiedfrom pituitaries of adult fish during the spawningseason. It is presumed, therefore, that this RIAmeasures the maturational GtH II(sLH) asdefined for salmon(Suzuki et al., 1988a,b). Hor-mone levels were expressed as the ratio betweenthe basal secretion and the secretion afterstimulation.

2.6. Breeding experiment

From the remaining fish, one male and threefemale fish from each treatment at a time werekept in breeding aquaria and observed for 3months. During this period, they were maintainedon the same diets as previously. Female fish killedby the males were immediately removed andreplaced as far as possible. All other conditions,such as water-quality parameters, light–darknessregime, etc., were also kept identical.

2.7. Statistical treatment

The deviation of the proportion of males andfemales from the expected ratio was tested by chi-square test. All other data were subjected to ANO-VA and statistical comparisons between treatmentswere made using Duncan’s multiple range test(Statistica for Windows, release 5.1 H, 1997 edi-tion). The significance of observed differences wastested atP-0.05.

3. Results

3.1. Feeding and growth

Fish in all groups consumed all of the feedprovided and did not show any abnormal behaviorduring the experimental period. In the S700yCtank, accidental blockage of the water inlet tuberesulted in the death of some fish. In other tanks,there were scattered deaths of one or two fishwhen they either jumped out of the aquaria orwere killed by other fish. Except for the accidentaldeaths in the S700yC tank mortality was isolatedand spread over the different treatments and nolinkage could be observed with any of the treat-ments. Dead fish were removed as soon as theywere noticed.The average final body weight of fish in each

treatment group is shown in Table 1. The averagebody weights of the treatment groups tended to be

higher than controls. Among the treatment groups,the fish that received the saponin-containing dietthroughout grew better than those that were put onthe control diet after 8 weeks. In both of thesegroups the fish which had once received or contin-ued to receive the S150 diet, had the highestspecific growth rate(Table 1). Whether the differ-ence in growth between different treatments wasstatistically significant could not be ascertainedsince all the fish in one aquarium were weighedtogether.

3.2. Sex ratio

The proportion of males and females in thetreatment groups is given in Fig. 1. There wereirregular sex ratios in the treatment groups com-pared to controls(Table 2). The number of maleswas higher in some of the groups that received thesaponin-containing diets. Only the S700 groupdeviated significantly from the expected 50:50ratio (Table 2). The control group had close to thetypical gender ratio.

3.3. Serum LH level

The measurements of LH level in the serum of5- and 6-month-old male and female tilapia didnot yield uniform results. The deviation in values


Fig. 1. Proportion of males and females(phenotypic) among the treatment groups at the end of 6 months.

in the individual groups(3 or 5 fish) were veryhigh, but there were still some differences amongtreatment groups(Table 3). However, no dietarysaponin-dependent trend or pattern of change inLH could be noticed. The hormone levels werehigher in males than in females at the age of 5months. The serum level in males increased two-to three fold from the level at 5 months to that at6 months. In the 6-month old female fish, the LHincreased several-fold during this interval, reachinglevels approximately double those of the 6-month-old males.

3.4. In vitro stimulation of LH secretion by QS

The effect of QS on taGtH release was examinedusing tilapia pituitary cells in primary culture.Exposing the cells for 5 h to QS resulted in amoderate increase in LH release similar to thatevoked by sGnRHa. However, the more purifiedQS (QS-P; 10, 100 or 200mgyml) evoked a dose-dependent increase in LH output which was sig-nificantly higher than that evoked by 10 nMsGnRHa(Fig. 2a). To determine whether QS canalter the cell response to GnRH, both substances

were added concomitantly. The selected saponinconcentration was 10mgyml to allow for anadditive effect. When QS and sGnRHa were addedsimultaneously, LH release was not significantlydifferent from basal, however, when QS-P andsGnRHa were added simultaneously, gonadotropinrelease was significantly higher than the basal- andGnRH-induced release(Fig. 2b). Exposure of thecells to 10mgyml saponin, in the presence of 20%FCS, abolished its stimulatory effect(Fig. 2c).

3.5. Serum and muscle cholesterol

There were no significant differences betweenserum cholesterol levels in the treatment groups.The cholesterol content in the serum of femalefish was approximately 50% higher than that ofmales. In both continuously saponin-fed and only-initially saponin-fed groups, the average serumcholesterol levels in males showed a steadilyincreasing trend, from C to S700yC or C to S700(Fig. 3). The coefficient of determination valuesof regression lines fitted to the average valueswere 0.62 and 0.69, respectively. No such trend


Table 2x andP values of the male–female distribution of tilapia in2

the various experimental treatments

Treatment Number* Number* Total x2 Pof males of females number

C 15 17 32 0.13 0.72C 17 14 31 0.29 0.59C 14 16 30 0.13 0.72C 15 15 30 0.00 1.0050yC 14 15 29 0.03 0.85150yC 15 12 27 0.33 0.56300yC 19 11 30 2.13 0.14500yC 16 13 29 0.31 0.58700yC 16 9 25 1.96 0.16S50 24 16 40 1.60 0.21S150 18 13 31 0.81 0.37S300 22 13 35 2.31 0.13S500 22 13 35 2.31 0.13S700 22 10 32 4.50 0.03

Each of the calculated chi-square values were comparedagainst the respectiveP-values to determine significant devi-ations from the expected 1:1 ratio. TheP-value is less than0.05 only in the case of the S700 group. C-tilapia fed controlfeed; S50yC to S700yC-tilapia fed diets containing 50,150....up to 700 ppmQuillaja saponins for 8 weeks(week 3to week 11) followed by control; S50 to S700-tilapia fed dietswith 50, 150...up to 700 ppmQuillaja saponins throughout.

Numbers also include fish that were sampled at 5 months*

and fish that died between the fifth month and the end of theexperiment.

Table 3Average serum LH levels(ngyml) of tilapia in treatment groups after 5(ns3 for males and females) and 6 (ns5 for males andfemales) months

Treatment Males Females

After 5 months After 6 months After 5 months After 6 months

Average SEM Average SEM Average SEM Average SEM

C 18.3abc 5.4 49.8abc 13.9 3.4bcd 1.3 142.6 55.6S50yC 29.6abc 0.4 60.7abc 54.2 16.1abc 10.4 107.4 39.4S150yC 9.4bc 5.2 36.7abc 21.3 3.1bcd 0.3 148.1 71.0S300yC 36.4abc 8.6 35.3abc 11.7 8.2abcd 2.4 70.7 19.1S500yC 51.7ab 14.6 36.0abc 11.2 3.8bcd 1.4 162.8 55.4S700yC 34.9abc 5.8 30.3abc 11.9 1.3cd 0.3 71.0 19.1S50 31.8abc 3.7 83.6abc 36.8 4.6bcd 1.8 201.1 63.3S150 15.3abc 12.8 85.0abc 25.2 20.6ab 9.1 148.4 81.0S300 34.9abc 24.8 36.1abc 24.0 6.0bcd 4.2 201.6 111.2S500 20.1abc 7.6 127.1ab 88.0 5.3bcd 2.3 111.8 63.7S700 32.2abc 22.7 15.1bc 5.8 3.5bcd 0.1 54.9 33.3

Averages not sharing common superscripts are significantly different(P-0.05). C-tilapia fed control feed; S50yC to S700yC-tilapiafed diets containing 50, 150....up to 700 ppmQuillaja saponins for 8 weeks(week 3 to week 11) followed by control; S50 to S700-tilapia fed diets with 50, 150...up to 700 ppmQuillaja saponins throughout.

existed in the average cholesterol values amongthe feeding classes of the female fish.The muscle cholesterol values did not differ

significantly among the treatment groups. The

average muscle cholesterol values in the femaleswere also higher than those of males, even thoughthe difference was not as big as in the case ofserum cholesterol. There were no apparent dietarytreatment-dependent trends among muscle choles-terol levels(Fig. 3).

3.6. Breeding trials

The dominant males in the S150yC, S500yCand S700yC groups were extremely aggressive andkilled all the supplied females within a very shorttime in the breeding tanks, and hence no obser-vations could be made on breeding in these groups.One female each in the S300 and S500 groupsspawned once immediately after being placed forbreeding, but the eggs did not hatch and were spitout by the females after 2 days; there was nofurther spawning for the following 3 months. Threefemale tilapia in the control group and one in theS300yC spawned after;4 weeks and producedfry. The mouth-brooding fish were removed fromthe breeding aquaria and were replaced as far aspossible.

4. Discussion

Dietary QS at a level of 3000 mg kg havey1

previously been reported to depress feed intakeand growth in Chinook salmon and rainbow trout(Bureau et al., 1998). Significant intestinal damagewas caused in both species at a dietary QS level


Fig. 2. The in vitro effect ofQuillaja saponin (QS) or purifiedQS (QS-P) on LH release from dispersed tilapia pituitary cells(LH denoted as ratio between basal secretion and secretionafter stimulation). (a) Cells were treated for 5 h with either100 nM salmon GnRH(GnRH) or with various concentrationsof the saponin(10, 100 or 200mgyml). (b) Release of LH bycultured tilapia pituitary cells stimulated by 100 nM salmonGnRH (GnRH), 10 mgyml QS, or a mixture of both com-pounds.(c) Dispersed tilapia pituitary cells were cultured inthe absence(basal) or presence of 20% fetal calf serum(ser),or with 10 mgyml QS (sap) or QS in the presence of serum(sapqser). Each value is the mean"S.E.M. of three differentwells from three or four independent experiments.

of 1500 ppm. In the current experiment, where thebody weight of tilapia increased from 30 mg to;100 g, QS did not cause suppression of feedingor any other apparent abnormal behaviour up to adietary level of 700 mg kg . The initial feedingy1

rate of 20% of body weight was higher than the

12–15% recommended by Shelton et al.(1981).The fish in the saponin-fed groups had higherabsolute specific growth rate(SGR) than controls.This was in agreement with our previous obser-vation of a growth promoting effect of dietary QSin tilapia (Francis et al., 2001). Oral applicationof other androgenic substances has also beenpreviously found to have a positive anabolic effectin tilapia (Tayamen and Shelton, 1978; Macintoshet al., 1988 also see review by Pandian and Sheela,1995), particularly at steroid doses that were sub-optimal for sex inversion. However, growth depres-sion occurred invariably over the long term(seereview by Pandian and Sheela, 1995). The averageSGR values of tilapia that received a saponin-supplemented diet throughout were higher thanthose receiving saponins only for the first 8 weeks.In both groups, maximum growth was achieved atthe 150 mg kg level of supplementation. As they1

fish in each aquaria were collectively weighedthere was no statistical treatment of the growthdata. In our previous experiment(Francis et al.,2001) tilapia fed 150 mg kg saponins hady1

significantly higher growth rates 3 weeks afterfeeding started. Tilapia fed 300 mg kg QSy1

showed a significantly higher body weight gainafter 14 weeks of feeding in that experiment.Results of the current experiment seemed to cor-roborate the growth-promoting effects of dietaryQS.The sex ratio of tilapia in the saponin-fed groups

deviated from the normal 50:50 ratio, with theS700 group showing a significant deviation fromthis pattern in favour of males. Deviation from thenormal sex ratio was also evident in the treatmentgroups that were fed saponins only for the first 8weeks, suggesting that sex-inversion occurs duringthis initial period. The first 21 days have beenjudged the most effective period for sex reversalin tilapia fry (Clemens and Inslee, 1968). Thepercentage of males in any of the treatments wasnot as high in the current experiment as in exper-iments that made use of synthetic androgens(Cruzand Mair, 1994; Gale et al., 1999). Even thoughthe concentration of the saponin mixture added tothe diet was much higher than the 60 mg kg ofy1

synthetic androgens used in most studies, theconcentration of saponins actually present waslower because the mixture used is known to haveonly approximately 20% of these compounds(approximate content of 10% sapogenin), theremaining 80% being made up of tannins and


Fig. 3. Cholesterol levels in the serum(mgy100 ml) and muscle(gy100 g) of male and female fish in the treatment groups. a and b,serum cholesterol levels of males; c and d serum cholesterol level of females; e and f, muscle cholesterol levels of males and females,respectively, in different treatments.

polyphenols(Kensil, 1996). Among the saponinsthemselves, there may be differences in effective-ness of the different compounds present. Thehighest proportion of males was found in the S700and S700yC groups. However, the growth-promot-ing effect of dietary QS was pronounced at the

much lower doses of 150 mg kg indicatingy1

potentially different mechanisms affecting the twoactions of dietary QS.Saponins have been previously reported to affect

the release of hormones, such as LH, from thepituitary (Benie et al., 1990). LH is considered to


regulate all aspects of teleost reproduction(Suzukiet al., 1988a) and is particularly important forfinal oocyte maturation and ovulation(Suzuki etal., 1988b). We previously observed the inhibitoryeffect of dietary QS on ovulation in female tilapiahaving apparently normal eggs in their ovaries(Francis et al., 2001; and unpublished observa-tions). It was, therefore, hypothesized that changesin LH levels would give an indication of themechanism of action of QS on tilapia at thepituitary level.We were able to demonstrate that QS promotes

LH release from cultured pituitary cells of tilapia.The more purified saponin was more efficient inincreasing the rate of hormone release indicatingthat saponins are the responsible active principles.Saponins fromPetersianthus macrocarpus havebeen shown to stimulate both FSH and LH releaseby cultured pituitary cells of rat(Benie et al.,1990). Saponin also promoted an increase in uter-ine weight and blocked the estrous cycle in theluteal phase. Although there is some evidence thatsaponin can act through permeabilization of cellmembranes, this seems at least not to be the onlymode of action in tilapia pituitary cells, since, inthe range of concentrations used, we found a dose-dependant response of LH to QS-P(Fig. 2). Wealso studied its effect on cultured cells in thepresence of serum. Cells incubated in the usualBSA-containing medium released LH in a dose-dependent manner. In contrast, when cells wereincubated with the same amount of saponin in thepresence of 20% FCS, the LH release was signif-icantly lower, and similar to the basal level. Sinceit is known that saponins have a high affinity forcholesterol and other lipids(see Fenwick et al.,1992), the serum protection may be due to sapo-nin’s adsorption by serum lipids, suppressing itsactivity. This effect of serum protection on gonad-otropin release from rat pituitary cells has alsobeen seen with another saponin fromP. macrocar-pus (El Izzi et al., 1992).The average LH values in vivo, however, did

not show any trends with respect to the effect ofdietary QS because of the extremely high variabil-ity within treatments. What was notable in thesemeasurements were the different values for the 5-and 6-month old tilapia. The increase in serum LHin females was particularly pronounced. This largedifference in LH levels may have been due to thefact that the tilapia entered the reproductive stageduring this period. During the recrudescence and

beginning of vitellogenesis stages LH levels in theplasma are very low while during final oocytematuration LH levels are quite high(Tacon et al.,2000; Rothbard et al., 1991). A reproduction cyclein tilapia takes 21–28 days. It is very likely thatduring the time of sampling at 5 months, the fishwere at the recrudescence stage, or too young toenter the reproduction stage, while at the age of 6months the fish were at the stage of final oocytematuration when LH levels are high.Since dietary QS was shown to affect the sex

ratio, it can be concluded that this change is notexerted via LH levels but rather via interferencein either the FSH at the pituitary level or thesteroids(probably androgens) at the gonadal level.The protective effect of the serum on the gonado-tropin release in vitro may not be as strong in vivodue to the low lipid concentration in tilapia serumcompared to 20% FCS.It has become increasingly clear that the lipid

environment of membrane proteins, including ionchannels, transporters and receptors, plays animportant role in their function(Bastiaanse et al.,1997). In biological membranes, cholesterol isorganized into structural and kinetic domains orpools. Saponins may interact with the polar headsof membrane phospholipids and cholesterolthrough their OH groups. Moreover, their hydro-phobic steroid backbone could intercalate into thehydrophobic interior of the bilayer. Both of theseeffects may contribute to altering the lipid envi-ronment around membrane proteins and activatespecific receptors, channels or enzymes(Attele etal., 1999). The observed saponin-induced dose-dependent LH release from tilapia pituitary cellsprovides evidence for this mode of action. More-over, the combination of natural GnRH and sapo-nin, both at sub-maximal doses, did not elicit ahigher response than each substance alone, sug-gesting that its mechanism of action may involveindirect activation of the receptor. Based on theirstructure, another suggested mode of action forsaponins is through binding to the estradiol recep-tor in mammals(Martin et al., 1978) and fish(Latonnelle et al., 2000). After binding to thereceptor, the saponin can elicit either agonistic orantagonistic effects in vivo and in vitro(Attele etal., 1999). The androgenic potency of QS in tilapiacan be explained by antagonistic effects on theestradiol receptor.The increasing trend in serum cholesterol con-

tent in male tilapia belonging to both saponin-fed


groups is in contrast to previous findings in whicha saponin-induced decline in serum cholesterolwas observed in gerbils and humans(Potter et al.,1993; Harris et al., 1997). There was also nodecline in the serum cholesterol content of femaletilapia in the saponin-fed groups relative to con-trols, but no dietary saponin-dependent trend wasin evidence. Moreover, muscle cholesterol levelswere not lower in the saponin groups compared tocontrols. The differences in serum and musclecholesterol contents between males and femaleswere also noteworthy.During the breeding trials, three female tilapia

belonging to the control group and one female fishbelonging to the S300yC group produced fry overa 3-month period. In the S500 and S700 groups,one female each produced eggs and were observedto mouth-brood immediately after they were placedtogether for breeding in the breeding aquaria. Butthey spit out the eggs after 2 days(similar tofemales having unfertilized eggs). The dominantmales(identifiable by their external reddish col-oration) in the S150yC, S500yC and S700yC wereespecially aggressive and killed all other fishplaced in the same aquaria. It has been previouslyreported that in trout fed a diet containing 500ppm genistein(a phytoestrogen structurally similarto saponin), testicular development was accelerat-ed in males, but spawning was delayed and gametequality impaired leading to a low percentage ofovulating females, a lower fertilisation rate and alower viability of fry (Bennetau-Pelissero et al.,2001). The results from the breeding experimentsneed to be treated as preliminary because of thelow number of females available in some of thesaponin-fed groups. Detailed trials, including arti-ficial fertilization methods, need to be carried outto further study the effects of dietary QS on thereproduction of Nile tilapia.In conclusion, dietary QS could potentially

replace hazardous synthetic androgens in produc-ing all-male populations or reducing female’s fer-tility if fed to tilapia at an early age, as is practisedwith synthetic androgen treatment.Quillajaextracts containing saponins are used commerciallyas flavourings and foam producers in a variety ofhuman food preparations(Kensil, 1996) and havebeen classified as ‘generally recognized as safe’(GRAS) in the US (Fenwick et al., 1992). Theoptimal concentration of QS for the production ofall-maleyinfertile female populations and its mech-anism of action need to be determined through

further studies. This optimal dosage could then befed to tilapia during the gonadal differentiationstage(up to 30 dpf forO. niloticus, Hines et al.,1999) of their life cycle.

Acknowledgments

The authors wish to thank Professor GabrieleHorstgen–Schwark, Institut fur Tierzucht und¨ ¨Haustiergenetik, Georg–August Universitat, Got-¨ ¨tingen, Germany for supplying tilapia fry. G. Fran-cis is thankful to the ‘Katholischer AkademischerAuslander Dienst’, Bonn, for providing a fellow-¨ship for Ph.D. studies in Germany.

References

Anken, R.H., Kappel, T.h., Slenzka, K., Rahmann, H., 1993.The early morphogenetic development of the Cichilid fishOreochromis mossambicus (Perciformes, Teleostei). Zool.Anz. 231, 1–10.

Attele, A.S., Wu, J.A., Yuan, C.S., 1999. Ginseng pharmacol-ogy: multiple constituents and multiple actions. Biochem.Pharmacol. 58, 1685–1693.

Bastiaanse, E.M., Hold, K.M., Van der Larse, A., 1997. Theeffect of membrane cholesterol content on ion transportprocesses in plasma mambrane. Cardiovasc. Res. 33,272–283.

Benie, -T., El-Izzi, -A., Tahiri, -C., Duval, -J., Thieulant, -M.L., 1990.Combretodendron africanum bark extract as anantifertility agent. I: Estrogenic effects in vivo and LHrelease by cultured gonadotrope cells. J. Ethno. Pharmacol.29, 13–23.

Bennetau-Pelissero, C., Breton, B.B., Bennetau, B., et al.,2001. Effect of genistein-enriched diets on the endocrineprocess of gametogenesis and on reproduction efficiency ofthe rainbow troutOncorhynchus mykiss. Gen. Comp. Endo-crinol. 121, 173–187.

Bogomolnaya, B., Yaron, Z., Hilge, V., Graesslin, D., Lichten-berg, V., Abraham, M., 1989. Isolation and radioimmuno-assay of a steroidogenic gonadotropin of tilapia. Isr. J.Aquacult.-Bamidgeh 41, 123–136.

Bureau, D.P., Harris, A.M., Cho, C.Y., 1998. The effects ofpurified alcohol extracts from soy products on feed intakeand growth of chinook salmon(Oncorhynchus tshawytscha)and rainbow trout(Oncorhynchus mykiss). Aquaculture 161,27–43.

Cheeke, P.R., 1996. Biological effects of feed and foragesaponins and their impact on animal production. In: Waller,G.R., Yamasaki, Y.(Eds.), Saponins Used in Food andAgriculture. Plenum press, New York, pp. 377–386.

Clemens, H.P., Inslee, T., 1968. The production of unisexualbroods ofTilapia mossambica sex-reversed with methyltes-tosterone. Trans. Am. Fish. Soc. 97, 18–21.

Cruz, E.V.M., Mair, G.C., 1994. Conditions for effectiveandrogen sex reversal inOreochromis niloticus (L.). Aqua-culture 122, 237–248.

El Izzi, A., Bennie, T., Thieulant, M.L., Le Men-Plivier, L.,Duval, J., 1992. Stimulation of LH release from culturedpituitary cells by saponins ofPetersianthus macrocarpus: apermeabilizing effect. Planta Med. 58, 229–233.


FAO 2001. FAO yearbook, Fishery statistics, Aquacultureproduction 1999. Vol 88y2.

Fenwick, G.R., Price, K.R., Tsukamoto, C., Okubo, K., 1992.Saponins. In: D’Mello, J.P.F., Duffus, C.M., Duffus, J.H.(Eds.), Toxic Substances in Crop Plants. The Royal Societyof Chemistry, London, UK, pp. 285–327.

Francis, G., Makkar, H.P.S., Becker, K., 2001. Effects ofQuillaja saponins on growth, metabolism, egg production,and muscle cholesterol in individually reared Nile tilapia(Oreochromis niloticus). Comp. Biochem. Physiol.(C) 129,105–114.

Francis, G., Makkar, H.P.S., Becker, K., 2002. Dietary supple-mentation with aQuillaja saponin mixture improves growthperformance and metabolic efficiency in common carp(Cyprinus carpio L.). Aquaculture 203, 311–320.

Gale, W.L., Fitzpatrik, M.S., Lucero, M., Contreras-Sanchez,W.M., Schreck, C.B., 1999. Masculinisation of Nile tilapia(Oreochromis niloticus) by immersion in androgens. Aqua-culture 178, 349–357.

Harris, W.S., Dujovne, C.A., Windsor, S.L., Gerrond, L.L.C.,Newton, F.A., Gelfand, R.A., 1997. Inhibiting cholesterolabsorption with CP-88,818(beta-tigogenin cellobioside;tiqueside): Studies in normal and hyperlipidemic subjects.J. Cardiovasc. Pharmacol. 30, 55–60.

Hines, G.A., Boots, L.R., Wibbels, T., Watts, S.A., 1999.Steroid levels and steroid metabolism in relation to earlygonadal development in tilapiaOreochromis niloticus(Teleostei: Cyprinoidei). Gen. Comp. Endocrinol. 114,235–248.

Jenkins, K.J., Atwal, A.S., 1994. Effects of dietary saponinson fecal bile acids and neutral sterols, and availability ofvitamins A and E in the chick. J. Nutr. Biochem. 5, 134–138.

Johnson, I.T., Gee, J.M., Price, K., Curl, C., Fenwick, G.R.,1986. Influence of saponins on gut permiability and activenutrient transport in vitro. J. Nutr. 116, 2270–2277.

Kensil, C.R., 1996. Saponins as vaccine adjuvants. Crit. Rev.Ther. Drug Carrier Syst. 13, 1–55.

Latonnelle, K., Bennetau-Pelissero, C., Fostier, A., Le Menn,F., 2000. Affinity of estradiol receptors from rainbow trouthepatocytes for phytoestrogens. In: Proceedings of the SixthInternational Symposium on the Reproductive Physiologyof Fish, Bergen, Norway, 4–9 July, 1999, p. 385.

Levavi-Sivan, B., Yaron, Z., 1992. Involvement of cyclicadenosine monophosphate in the stimulation of gonadotro-pin secretion from the pituitary of the teleost fish, tilapia.Mol. Cell. Endocrinol. 85, 175–182.

Levavi-Sivan, B., Ophir, M., Yaron, Z., 1995. Possible sites ofdopaminergic inhibition of gonadotropin release from thepituitary of a teleost fish, tilapia. Mol. Cell. Endocrinol.109, 87–95.

Macintosh, D.J., Singh, T.B., Little, D.C., Edwards, P., 1988.Growth and sexual development of 17a-methyltestosteroneand progesterone-treated Nile tilapia(O. niloticus) reared inearthen ponds. In: Pullin, R.S.V., Bhukaswan, T., Tonguthai,K., Maclean J.L.(Eds.), The Second International Sympo-sium on Tilapia in Aquaculture, Bangkok, Thailand, 16–20March 1989. Department of Fisheries, Bangkok, Thailandand ICLARM, Manila, Philippines, ICLARM conferenceproceedings, Vol. 15, pp. 457–463.

MacKenzie, D., November 1996. Doctors farm fish for insulin.New Scientist 16, 20.

Martin, P.M., Horwitz, K.B., Ryan, D.S., McGuire, W.L., 1978.Phytestrogen interaction with estrogen receptors in humanbreast cancer cells. Endocrinology 103, 1860–1867.

Pandian, T.J., Sheela, S.G., 1995. Hormonal induction of sexreversal in fish. Aquaculture 138, 1–22.

Petit, P.R., Sauvaire, Y., Ponsin, G., Manteghetti, M., Fave,A., Ribes, G., 1993. Effects of a fenugreek seed extract onfeeding behaviour in the rat: metabolic endocrine correlates.Pharmacol. Biochem. Behav. 45, 369–374.

Potter, S.M., Jimenez-Flores, R., Pollack, J., Lone, T.A.,Berber-Jimenez, M.D., 1993. Protein saponin interaction andits influence on blood lipids. J. Agric. Food Chem. 41,1287–1291.

Quin, G.-W., Xu, R.-S., 1998. Recent advances in bioactivenatural products from Chinese medicinal plants. Med. Res.Rev. 18, 375–382.

Riguera, R., 1997. Isolating bioactive compounds from marineorganisms. J. Mar. Biotechnol. 5, 187–193.

Rothbard, S. Ofir, M., Levavi-Sivan, B., Yaron, Z. 1991.Hormonal profile associated with breeding behavior inOreochromis niloticus. In: Scott, A.P., Sumpter, J.P., KimeD.E., Rolfe M.S.(Eds.), Reproductive Biology of Fish—Proceedings of Fourth Symposium on Reproductive Physi-ology of Fish, FishSymp. 91, Sheffield, p. 206.

Shelton, W.L., Rodriguez-Guerrero, D., Lopez-Macias, J.,1981. Factors affecting androgen sex reversal ofTilapiaaurea. Aquaculture 25, 59–65.

Shimoyamada, M., Ikedo, S., Ootsubo, R., Watanabe, K.,1998. Effects of soybean saponins on chymotryptic hydro-lyses of soybean proteins. J. Agric. Food Chem. 46,4793–4797.

Southon, S., Johnson, I.T., Gee, J.M., Price, K.R., 1988. Theeffect ofGypsophylla saponins in the diet on mineral statusand plasma cholesterol concentration in the rat. Br. J. Nutr.59, 49–55.

Suzuki, K., Kawauchi, H., Nagahama, Y., 1988a. Isolation andcharacterization of two distinct gonadotropins from chumsalmon pituitary glands. Gen. Comp. Endocrinol. 71,292–301.

Suzuki, K., Kawauchi, H., Nagahama, Y., 1988b. Isolation andcharacterization of subunits of two distinct gonadotropinsfrom chum salmon pituitary glands. Gen. Comp. Endocrinol.71, 302–306.

Tacon, P., Baroiller, J.F., Le Bail, P.Y., Prunet, P., Jalabert, B.,2000. Effect of egg deprivation on sex steroids, gonadotro-pin, prolactin, and growth hormone profiles during thereproductive cycle of the mouthbrooding cichlid fishOreo-chromis niloticus. Gen. Comp. Endocrinol. 117, 54–65.

Tewary, P.V., Chaturvedi, C., Pandey, V.B., 1973. Antifertilityactivity of Costus speciosus Sm. Indian J. Pharm. 35,114–115.

Tayamen, M.M., Shelton, W.L., 1978. Inducement of sex-reversal ofSarotherodon niloticus (Linnaeus). Aquaculture14, 349–354.

Yoshiki, Y., Kudou, S., Okubo, K., et al., 1998. Relationshipbetween chemical structures and biological activities oftriterpenoid saponins from soybeanwReviewx. Biosci. Bio-tech. Bioch. 62, 2291–2299.

Documents

Effects of long term feeding of Quillaja saponins on sex ratio, muscle and serum cholesterol and LH levels in Nile tilapia (Oreochromis niloticus (L.))