7152-15539-1-SM

Indian J. Fish., 53(3) : 283-290, Jul.-Sep., 2006
283
Effect of dietary administration of
methyltestosterone on the growth and sex
reversal of two ornamental fish species
R. JAMES AND K. SAMPATH
Department of Zoology, V.O. Chidambaram College, Tuticorin - 628 008, Tamilnadu,
India
ABSTRACT
Efficacy of androgenic hormone, methyltestoterone (MT) on growth, sex reversal
and reproductive performance of two ornamental fishes, red swordtail
Xiphophorus helleri and siamese fighting fish Betta splendens were investigated.
30 day old juveniles were fed ad libitum on diets containing different levels of
MT (0,10,20,40 and 60 mg kg-1 diet) during the experimental period. The mean
body weight and length and specific growth rate of X. helleri and B. splendens
were higher in the MT 10 mg kg-1 treated groups than other levels of MT treated
groups, and hence 10 mg kg-1 is considered as optimum dose for attaining higher
growth rate. Sex determination of the fishes based on the secondary sexual
characteristics showed increased percentage of males with higher dose levels of
MT-treated groups. B. splendens treated with 20,40,60 mg kg-1 produced 100%
male population on day 84,56 and 56, respectively, while X. helleri treated with
40 and 60 mg kg-1 produced 100% male population on day 70 and 56.
Introduction
Studies on the application of steroid
hormones in fish have drawn the attention of aquaculturists and researchers to
control unwanted reproduction and divert energy for somatic growth in the economically important edible fishes
(Monfort, 1974; Nirmala and Pandian,
1983; Sindhu and Pandian, 1984). The
technique of sex manipulation by hormone treatment is also of interest to the
aquarists as the price of some species of
aquarium fish depend on their sex. Males
of most of the ornamental fishes are colourful and have a higher commercial
value than females. In Poeciliidae, male
red swordtail Xiphophorus helleri is more
attractive than female due to their long
sword (1 or 2) behind the caudal fin on
the ventral side. In Anabantidae, the
siamese fighting fish, Betta splendens
male has attractive colour and large fins.
The most commonly employed technique
for monosex production is administration
of steroids to undifferentiated fish
through diets. Many successful attempts
have been made in edible fishes to produce monosex by sex reversal using selected steroids (Shelton et al., 1978;
Pandian and Varadaraj, 1988; Komen et
al., 1989). Age, size of animals, hormone
dose and the duration of hormone exposure are major factors determining the
success of sex reversal in fish and these
R. James and K. Sampath
vary from species to species (Nakamura,
1984; James, 1998). The present study
has been undertaken to study the effect
of male steroid hormone methyltestosterone on growth, sex reversal and reproduction in the aquarium fishes, red
swordtail Xiphophorus helleri and
siamese fighting fish, Betta splendens.
Materials and methods
Three hundred and seventy five active juveniles of 30 day old X. helleri (13.2
+ 1.17 mm; 50+5 mg) were separately
collected from the laboratory-bred brooders. The fishes were divided into five
groups and treated with five different
doses (0,10,20,40 and 60 mg kg-1 diet) of
male steroid hormone, methyltestosterone (MT). Each group consisted of 25 individuals of homogenous body size and
they were cultured in circular cement
aquaria (1.75’x1.50’;110 l capacity). The
experimental test fish X. helleri was
maintained in 90 l of water and the obligate air-breathing fish B.splendens in 50
l of water. The tanks were filled with
dechlorinated well water and 75% of the
water was changed twice in a week. The
water quality parameters were estimated
following the method of Degani et al
(1985). The average hydrological parameters were : temperature : 28.5+1°C; pH:
7.7+0.2; salinity: 0.11+0.01 ppt and DO :
4.39+0.21 ml l-1; water hardness : 316 mg
CaCO3l-1.
Medically available tablets (Mixogen
- Organon company) were utilized as the
source of MT. MT was incorporated in the
diets by spraying the hormone dissolved
in 95% ethanol on to cooked and cooled
feed ingredients containing 35% protein.
MT was administered at five different
levels viz. 0,10, 20, 40 and 60 mg kg-1 diet
by dissolving appropriate amount of hormone tablets in 95% ethanol. The feed
ingredients were thoroughly mixed and
extruded through a mechanical pelleti-
284
zing machine having a perforation diameter of 2 mm. The pellet was sun dried
till the moisture content was reduced to
less than 10%. The control diet was also
prepared in the same way, without the
addition of hormone.
The first group of X. helleri and B.
splendens were fed with hormone free
35% protein (control) diet. The second,
third, fourth and fifth groups of both species were fed with diets containing 10,
20, 40 and 60 mg MT kg-1, respectively.
Feeding was done once in the morning
(at 0800 hr) by placing the required and
known quantum of diet in a petridish in
each tank. Uneaten feed was removed
after 2 hours of feeding and dried in a
hot air oven at 800C. Once in 2 weeks,
the weight of the experimental fishes was
measured and the amount of feed given
was readjusted according to the weight
(4% of body weight) of the fishes. All the
experimental fishes were sampled at
every 28 days interval from 0 to 98 days
and the mean body length, weight and
selected feeding parameters were measured. Two females from each replicate
tank were sacrificed simultaneously and
ovaries dissected out and weighed. From
these weights, the gonadosomatic index
(GSI) was computed following Dahlgren
(1979).
Gonadosomaticindex (%) = Wet weight of gonad x 100
Wet weight of fish
Mean body weight (g) was calculated
by dividing the wet weight of test animals by total number of animals in the
aquarium. ‘Sacrifice method’ was
adopted to estimate growth (Maynard
and Loosli, 1962) of fish. Fish, feed samples, unconsumed feed and ovary were
weighed in an electrical monopan balance
to 0.1 mg accuracy. The following formulae were used for the estimation of the
chosen feeding parameters.
Methyltestosterone on growth and sex reversal in two ornamental species
Feed consumption = Total feed offered (g dry matter)
Quantity of
unconsumed feed
Gain in weight =
(g dry matter)
Final dry weight Initial dry weight
Daily specific growth rate (SGR) for
weight and length, were calculated using the following formulae.
SGR (% weight day-1) = (Wt1-Wt0) x 100
t
-1
SGR (% length day ) = (Lt1-Lt0) x 100
t
where Wt0 and Lt0 and Wt1 and Lt1
are weights and lengths of the fishes
prior to commencement and termination
of the experiment respectively. Data on
growth and reproduction were analysed
by using t-test (Zar, 1974).
The sex ratio was measured at every
14 days intervals from 0 to 98 days when
both species began to brood. The sex was
identified based on the secondary sexual
characteristics of the fish. In X. helleri,
fish with long gonopodium and elongated
lower base of the caudal fin (sword) were
identified as males, while those without
these features were regarded as females.
In B. splendens, fishes having a long flowing fin with vibrant colouration were
grouped as males and fishes of dull colour with shorter fins as females. When
X. helleri attained sexual maturity on day
98 (MT 0, 10 and 20 mg kg-1), randomly
chosen two female broods were reared
with a male in the same tank with sufficient quantity of macrophyte, Hydrilla
species till the completion of the first
breeding. Female broods of swordtail released young ones, which were isolated
from their parents and then counted.
After sexual maturity on day 98 (MT 0
and 10 mg kg-1) a male B. splendens was
paired with a female in a plastic through
285
containing 5 l of water for spawning. Few
plant tufts were placed on the surface to
provide shelter for the female against the
highly aggressive male. Eggs were collected and counted. Unhatched eggs were
also counted after 36-40 h of incubation
by males.
Results
X. helleri and B. splendens treated
with 10 mg kg-1 MT recorded the highest
growth while 0, 20, 40 and 60 mg kg-1
MT treated groups showed significantly
(P < 0.05) less growth. The specific
growth rate for weight and length of two
species was higher in the 10 mg kg-1 MT
group than those treated with 0, 20, 40
and 60 mg kg-1 MT. Similar result was
obtained for feed consumption and gain
in weight also (Table 1 and 2).
Gonad weight and gonadosomatic
index decreased with increase in their dietary dose of MT from 0 to 20 mg kg-1 in
X. helleri and 0-10 mg kg -1 MT in B.
splendens. X. helleri fed with the control
diet released 32 fry, while fish fed with
MT 10 mg kg-1 diet released 19 (t=2.55;
P<0.05) fry only; however, test fish
treated with MT 20 mg kg-1 did not breed
during the experiment (Table 3). On the
contrary, the number of eggs spawned
was more in B. splendens fed with MT
10 mg kg-1 diet than control. Fishes fed
on MT 10 mg kg-1 diet produced more
males in their first generation; however,
in control treatment, female population
was slightly higher. Cent per cent male
population was obtained when X. helleri
was fed with MT 40 and 60 mg kg-1 diets
and in B. splendens, all male population
was obtained at MT 20,40 and 60 mg
kg-1 diets and it was time-and dose-dependent. For instance, B. splendens
treated with MT 20, 40, and 60 mg kg-1
produced 100% male population on day
84, 56 and 56 respectively. However, X.
helleri treated with MT 40 and 60 mg
286
R. James and K. Sampath
TABLE 1 : Effect of methyltestosterone at different levels on the mean body weight and length
and specific growth rate of Xiphophorus helleri over a period of 98 days. Each value is the
mean (X+SD) of three observations.
Parameters
MT-concentrations (mg kg-1)
0
10
20
40
60
0.05+0.001
0.05+0.021 0.05+0.014
0.05+0.014
Average initial
0.05+0.004
weight (g)
1.14+0.07
0.71+0.06
0.36+0.03
0.19+0.02
Average final
0.40+0.04
weight (g)
1.09+0.04
0.66+0.03
0.31+0.01
0.14+0.00
Net gain in
0.35+0.01
weight (g)
0.36+0.01
1.11+0.11
0.67+0.03
0.44+0.03
0.25+0.01
SGR (% W day-1)
13.2+1.17
13.2+1.17
13.2+1.17
13.2+1.17
Average initial
13.2+1.17
length (mm)
45.2+3.35
36.0+1.87
32.6+2.10
25.6+1.82
Average final
34.4+2.45
length (mm)
32.0+1.63
22.8+2.10
19.4+1.40
12.4+0.65
Net gain in
21.2+1.16
length
22.0+1.50
33.0+1.78
23.0+1.10
28.0+1.21
22.0+1.22
SGR (% L day-1)
Food consumed
16.67+1.70
34.21+1.60
15.99+1.10 14.52+0.80
6.01+0.40
g dry weight)
5.12+0.40
2.95+0.20
1.67+0.10
0.89+0.10
Gain in weight
1.62+0.10
(g dry weight)
TABLE 2 : Effect of methyltestosterone at different levels on the mean body weight and length
and specific growth rate of Betta splendens over a period of 98 days. Each value is the mean
(X+SD) of three observations.
Parameters
MT-concentrations (mg kg-1)
0
10
20
40
60
0.07+0.014
0.06+0.021 0.06+0.028
0.07+0.017
Average initial
0.06+0.008
weight (g)
1.68+0.04
0.53+0.04
0.25+0.22
0.18+0.20
Average final
0.35+0.03
weight (g)
1.61+0.03
0.47+0.02
0.19+0
0.11+0
Net gain in
0.29+0.01
weight (g)
0.35+0.02
1.73+0.01
0.56+0.02
0.34+0.01
0.20+0.01
SGR (% W day-1)
14.8+0.5
14.8+0.5
14.8+0.5
14.8+0.5
Average initial
14.8+0.5
length (mm)
48.9+2.27
36.7+2.28
33.4+2.65
32.6+3.10
Average final
31.9+2.85
length (mm)
34.1+2.14
21.9+1.42
18.6+0.75
17.8+1.30
Net gain in
17.0+1.04
length
20+1.50
41+2.40
26+1.06
33+2.10
32+2.33
SGR (% L day-1)
Food consumed
6.86+0.33
12.68+0.46
9.99+0.85
3.58+0.11
2.53+0.18
g dry weight)
3.0+0.04
2.25+0.42
0.90+0.05
0.54+0.03
Gain in weight
1.26+0.03
(g dry weight)
kg-1 produced 100% male population on
day 70 and 56. Cent per cent male population was obtained in B. splendens
(oviparous) at low dosage and earlier
than X. helleri (ovoviviparous). In the
control group, male:female ratio aver-
aged to 37.5:62.5% in both species.
Discussion
The dietary administration of MT at
10 mg kg-1 elicited the maximum growth
rate in X. helleri and B. splendens, while
Methyltestosterone on growth and sex reversal in two ornamental species
287
TABLE 3 : Effect of different levels of methyltestosterone on gonad weight, gonadosomatic index
and sex ratio in Xiphophorus helleri and Betta splendens. Each value is the mean (X+SD)
performance of six observations.
Parameters
MT-concentrations (mg kg-1)
0
10
20
40
60
Xiphophorus helleri
Ovary
Gonad wet weight (mg)
Gonadosomatic index (%)
Fertility
No. of days taken for breeding
No. of young ones released
/bred
Sex ratio (%)
Male
Female
9.5+0.45
8.5+0.73
4.5+0.16
1.50+0.04
1.38+0.10
0.89+0.03
144+9
32+6
149+11
19+4
Not bred
Not bred
-do-do-
38+4
62+5
86+8
14+2
Not bred
Not bred
-do-do-
All individuals were
transformed into
males
-do-
Betta splendens
Ovary
Gonad wet weight (mg)
Gonadosomatic index (%)
Fecundity
No. of eggs spawned
No. of eggs hatched
Egg hatchability (%)
Sex ratio (%)
Male
Female
87+4.8
75+6.0
17+1.4
13.2+1.1
297+18
269+20
90.6+6.2
364+24
338+19
92.86+5.2
-do-do-do-
46+3.5
54+2.1
78+6.2
22+0.7
-do-do-
20 to 60 mg MT reduced the growth rate.
It suggests that MT has anabolic effect
at lower dose stimulating feed intake and
growth and catabolic effect at higher
doses causing reduction in growth. Similar observation was made in young ones
of the platy, Xiphophorus maculatus fed
with 17 a-methyltestosterone (Felix,
1989). Basavaraja et al. (1989) reported
that lower dose (2.5 ppm) decreased the
growth rate in the carp, Cyprinus carpio.
Growth reduction in X. helleri and B.
splendens at higher doses of MT (20-60
mg kg-1) may be due to the catabolic actions as reported by Lone and Matty
(1983) in the common carp. Donaldson
et al. (1979) reported that higher doses
All individuals were
transformed into males
-do-
of some steroid hormones exert deleterious effect on various organs and cumulatively cancel the growth promoting effects. Steroid hormones 17 a- MT and 17
b-estradiol at higher doses (50-75 m/g fish
weight) did not accelerate growth in juvenile X. helleri (Lim et al., 1992).
In the present study, gonad weight
and gonadosomatic index of experimental animals decreased with an increase
of hormone dose beyond the optimum
dosage and it negatively reflected on the
reproductive performance (Table 3). Van
den Hurk and Slof (1981) found that
higher doses of steroid hormones have inhibitory effect on gonad development in
Salmo gairdneri.
R. James and K. Sampath
288
Fig. 1. Impact of methyltestosterone on sex ratio (% male or female) in Xiphophorus helleri
and Betta splendens as a function of treatment period.
Methyltestosterone on growth and sex reversal in two ornamental species
The present study reveal that MT
has gradually changed the undifferentiated 30 days old X. helleri and B.
splendens into all male populations and
the change was time-and dose-dependent (Fig.1) The production of all male
population in short duration would reduce the feed cost and the labour involved
and simultaneously enhance substantial
savings in production cost. NavaBautista and Rodriguez-Gutierrez (1997)
found that 35 mg kg-1 17 a-MT, produced
100% masculinization in X. helleri after
20 days of hormone treatment.
Kavumpurath and Pandian (1994) reported that 15 mg kg-1 17 a MT produced
100% masculinization in B. splendens
and these values are close to the present
study (20-40 mg kg-1). Lim et al. (1992)
found that 100% masculizination was
achieved in X. helleri when it was treated
with high doses of 17 a-MT (500-750 mg
g-1). Medically available MT is cheap and
has no side effects, while 17a-MT is costly
and exhibit the catabolic and side effects
at low doses.
Cent per cent masculinization was
achieved in B. splendens (oviparous) at
low doses and earlier than X. helleri (ovoviviparous) when treated with MT
(Fig.1). Pandian (1993) found that oviparous cichlids and anabantids require
minimum dose of MT, 5 and 50 mg kg-1
and brooding poeciliids (ovo-viviparous)
require higher doses of MT (300 to 500
mg kg-1) for the production of 100% male.
Lim et al. (1992) reported that sex reversal in poeciliids have not been completely successful, since they are ovo-viviparous and the sex of the fry is fixed in
early stages. According to Yamazaki
(1993), sex is fixed during embryonic
stages of poeciliids and MT should be
given to gravid females to get all male
individuals.
In conclusion, X. helleri and B.
289
splendens fed with optimum dosage of
MT (10 mg kg-1) elicited higher growth
than those treated with higher does of
MT eventhough higher doses of MT produced all monosex populations. Production of monosex populations is of great
importance in aquaculture in order to
overcome early reproduction and indiscriminate mating which results in
stunted growth and poor quality for traits
which are of importance in ornamental
fish. Stocking with monosex populations
ensures better growth of the fish since
the energy wasted on gonadal maturation is channeled to protein production
and thus yields higher fish production.
This is also an important method to produce all male or all formal populations,
where a particular sex is preferred for
ornamental purposes (Van Den Hurk and
Slog, (1981).
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