The lsraeliJournal of Aquaculture - Bamidgeh 4O(l),
lgg4,
g-9.
INVIABILITY OF YY ZYGOTES OF THE FIGHTING FISH,
BETTA SPLENDENS
Thomas George, Thavamani Jegajothivel Pandian and Soosamma Kavumpurath
School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, lndia
(Received 30.5.93, Accepted 7.9.93)
Abstract
Feminizdtion was achieved in the fighting fish, Beffa splendens, by treating 3-day old fry with
20 mg diethylstilbesterol, 50 mg 17 cr-ethynylestradiol or 125 mg B-estradiol per kg food for 40
days. Sex reversed heterogametic females (XY) were identified by progeny testing. Eggs collected from these females were activated with UV irradiated Oreochromis mossambicus sperm
and subjected to pressure shock at 7000 psi for 6 min to induce diploidy. However, the YY
males of B. splendens were not viabte and succumbed before hatching.
.
Introduction
The fighting fish, Betta splendens, is a
popular aquarium fish, appearing in a wide
range of beautiful colors. The males of this
species are well known for their aggressive
display. Therefore, maintenance and breeding
of male populations has generated a great
amount of commercial interest. lt is now possible to produce monosex male fighting fish
by rearing 3-day old fry on an androgensupplemented diet, strictly adhering to the critical minimum dosage and duration (Kavumpurath and Pandian, 1992b, 1993a). An alternate technique for commercial production of
all-male populations is the production of YY
males. Sperm from YY males can be used to
fertilize eggs from normal (XX) females for
mass production of all-male populations. The
techniques of endocrine sex reversal at the
G1 generation and selective breeding at the
G2 generation have been used in Oryzias
latipes (Yamamoto, 1955), Carassius auratus
(Yamamoto, 1975) and Poecilia reticulata
(Kavumpurath and Pandian, 1992a) to produce viable YY males. lnviability of certain YY
genotypes has been repofted in O. latipes
(Yamamoto, 1964).and salmonids (Hunter et
George et al.
al., 1982; Parsons and Thorgaard, 1985). Endocrine sex reversal and selective breeding
require progeny testing, identification of heter-
ogametic and homogametic individuals and
subsequent breeding. Scott et al. (1989) and
Varadaraj and Pandian (1989) have described an alternate technique for "supermale" production (endocrine sex reversal and
gynogenesis), which does not require long
term rearing (to maturation) and progeny testing. The present study was carried out to de-
termine the viability of YY male fighting fish
produced by integrating the techniques of sex
reversal and gynogenesis.
Materials and Methods
Three day old fighting fish fry were treated
with B-estradiol (125 mdkg food),
17a-
ethynylestradiol (50 mg/kg food), or diethylstilbesterol (20 mg/kg food) lor 4O days (Kavumpurath and Pandian, 1993a). The steroid sup-
amount of feed and stirred thoroughly. The sol-
vent was allowed to evaporate from the feed
by air drying in darkness. Control feed was
prepared in the same manner by treating the
feed with the same volume of ethanol but without the hormone. The fish were fed the hormone supplemented feed ad libitum three
times a day during the treatment. Treated fry
were reared in 30 | tanks until they were sexu-
ally mature. These were individually mated
with normal males, and sex-reversed heterogametic females (XY) were identified by the
sex ratio of their progeny. The heterogametic
females were used for YY male production following the protocol shown in Fig. 1. Eggs collected from sex-reversed and normal females
were activated with UV irradiated tilapia (Oreochromis mossamb,rbusl sperm and, 2.5 min
after fertilization, subjected to
a
pressure
plemented feed was prepared using the
shock of 7000 psifor 6 min to cause diploidization as described by Kavumpurath and Pandian (1993b).
alcohol evaporation method (Guerrero, 1975).
A stock solution of the hormone was prepared
by dissolving the steroid in ethanol or acetone
at a concentration of 1 mg/ml. The stock solution was diluted to the desired level in 95%
The results of progeny testing hormone
treated females are presented in Table 1.
Among the B-estradiot treated group, 4 te-
distilled ethanol, sprayed over
Go
YX
a
a
weighed
Results
males were identified as heterogametic on the
0XY
.7
6-
U)
-c
Go
XX Xy I
UV irradiated sperm
c
o
1218
Gz
XX
ad
(50%)
-
YY
(50%)
Fig. 1. Production of YY male Betta splendens by integrating endocrine sex reversal and gynogenesis.
lnviability of YY zygotes of the fighting fish, Betta splendens
Table 1. Results of progeny testing ol Betta splendens females, produced by treatment with
B-estradiol (A1-Ag), 17cr-ethynylestradiol (B1-B10) or diethylstilbesterol (C1-C8) and mated with
normal XY males.
Female parent
Total offspring
tested
(no.)*
.
Sex ratio
(Female
:
Male)
lnferred
genotype of
Go parent
A1
241
0.9
XX
A2
254
0.9
XX
A3
215
2.4
XY
A4
195
2.5
XY
A5
202
1.0
XX
A6
151
1.9
XY
A7
115
2.4
XY
A8
126
1.0
XX
A9
147
1.0
XX
B1
262
2.1
XY
?2
250
1.0
XX
B3
165
1.6
XX
B4
183
0.9
XX
B5
139
1.0
XX
B6
121
0.8
XX
87
253
2.3
XY
B8
251
2.1
XY
B9
180
0.9
XX
810
141
0.8
XX
c1
147
2.3
XY
C2
263
1.0
XX
c3
c4
241
1.0
XX
143
0.9
XX
cs
c6
c7
c8
124
2.4
XY
141
2,5
XY
238
1.0
XX
166
1.7
XX
Offspring were pooled from 2-3 separate matings.
George et al.
Table 2. Survival and sex distribution of diploid gynogens produced trom Befta splendens
treated with selected steriods.
Female
parent
Eggs
used Hatched Abnormal Suruivalto Animal Sex distribution
feeding stage sexed
(no.)
Male Female
f/,)
p-estradiol
A1
113
65
9
48
33
M
128
66
15
44
40
A3.
162
36
17
27
22
A4*
97
29
20
21
16
0
0
0
0
A5
135
64
7
5B
47
o
A6.
105
35
21
24
29
A7*
146
37
23
25
u
A8
118
55
14
53
39
67
10
47
.92
A9
100
100
100
100
i.
1oo
31
0
694
0
0
100
100
100,
$
lTii-estradiot
81'
210
B3
84
36
29
23
31
0
100
116
53
25
44
36
0
100
119
53
13
41
30
0
100
B5
152
70
11
55
49
0
100
B6
98
54
,7
48
25
0
100
87*
139
33
12
23
19
0
100
Bg.
194
38
29
23
19
0
100
{
a
B9
187
69
14
42
54
0
100
810
187
59
14
42
54
0
100
Diethylstilbesterol
*
c1.
163
30
19
21
37
100
c2
204
65
14
44
52
100
c3
148
57
17
41
41
100
C4
136
57
11
43
39
100
c5*
184
36
20
24
32
100
c6.
154
36
20
26
33
100
c7
173
71
12
51
62
100
c8
97
5
12
44
21
100
"
Sex reversed XY females.
lnviability of YY zygotes of the fighting fish, Betta splendens
basis of the sex ratio of their progeny. In the
17 o-ethynylestradiol and
diethylstilbesterol
treated groups there were 6 heterogametic females, 3 in each group. All the females, both
heterogametic and homogametic, were subjected to gynogenesis and all, except one
(A7), produced all-female offspring (Table 2).
One heterogametic female produced
60/0
males, all of which succumbed before reaching sexual maturity. There was a remarkable
difference in the hatching and survival of the
gynogenetic fry produced by the homogametic and the heterogametic females. Survival of
fry from homogametic females ranged
be-
tween 41Y" and 55o/", whereas that from heterogametic females was only 21-27o/o, sug-
gesting that sex dependent
mortality
occurred. Evidently, YY males produced by
gynogenesis of sex-reversed females suc-
only two of the 21 animals tested were
yy
males. Survival of homogametic males (yy) is
far lower than that of normal heterogametic
males (Pandian et al., 1993). lt appears that
the viability of YY genotypes varies from species to species. The present study records evidence for the inviability of YY zygotes in fighting fish. ln the case of YY male production
through endocrine sex reversal and gynogenesis, both Y chromosomes are derived from
the mother. lt has not yet been tested whether
the YY individuals would survive if their y
chromosomes were derived one from the
mother and the other from the father. Therefore, production of YY males through sex reversal and selective breeding is to be tested
further and more research has to be carried
out to confirm the viability of YY zygotes in B.
splendens.
cumbed before hatching.
Acknowledgments
Discussion
ln B. splendens it is possible to obtain estrogen induced XY females in abundance and
to determine the viability of YY zygotes. Theoretically, one would expect 50% males (YY)
and 50% females (XX) from gynogenesis of
sex-reversed females (Varadaraj and Pandian, 1989). The fact that the eggs of thO sex-
reversed females (XY) suffered relatively
higher mortality suggests inviability of YY
males. The YY genotype seems
to
render
some sort of lethality in the zygotes. The occurrence of 6"/" males in the progeny of one
female may be explained by the possibility
that they were immature females or produced
by experimental error. The viability of the YY
zygote is a challenging problem. For instance,
Winge (1934) and Winge and Ditlevsen
(1938) demonstrated viability of Yma YPa but
lethality o1 ymayma in the guppy. Winge and
Ditlevsen (1938) assumed that a lethal factor
in the Y-chromosome in a homozygous condition was responsible for the death of the fish.
Although Yamamoto (1955) produced viable
O. latipes, the rarity of surviving YY zygotes is
reflected in his experiments. The actual proportion of XY to YY significantly deviated from
the expected 2:1 ratio. Of 57 males, singly
tested by mating, only two proved to be viable
YY males. Kavumpurath and Pandian (1992a)
produced YY males in Poecilia reticulata, but
Financial support received from the CSIR
'and UGC, New Delhi, is gratefully acknowledged.
References
Guerrero, R.D., 1975. Use of androgen for
the production of all-male Tilapia
(Steindachner). Trans.
aurea
Am. Fish. Sci., 104:
342-348.
Hunter, G.A., Donaldson, E.M., Goetz, F.W.
and P.R. Edgell, 1982. Production of all female and sterile group of coho salmon (Oncorhyncus kisutch) and experimental evidence
for mafe heterogamety. Trans. Am. Fish. Sci.,
111:367-372.
Kavumpurath, S. and T.J. Pandian, 1gg2a.
Production of YY male guppy Poecilia reticulata by endocrine sex reversal and progeny
testing. J. Asian Fish. Sci.,5:265-276.
Kavumpurath, S. and T.J. Pandian, 1gg2b.
The development of all male sterile triploid
fighting tish (Betta splendens Regan) by integrating hormonal sex reversal of broodstock
and chromosome-set manipulation. lsraeli J.
Aquacult. - Bamidgeh, 44:11 1-1 1 9.
Kavumpurath, S. and T.J. Pandian, 1993a.
Determination of labile period and critical
dose for sex reversal by oral administration of
estrogens tn Betta splendens (Regan). lndian
J. Exp. Biol., 31:16-20.
Kavumpurath, S. and T.J. Pandian, 1gg3b.
George et al.
Induction of heterozygous and homozygous
diploid gynogenesis in B. splendens (Regan)
Management, tn press.
technique. Curr. Scr., 58:434-441.
Winge, O., 1934. The experimental alteration
of sex chromosomes and vice versa, as illustrated by Lebistes. C.R. Trav. Lab. Carlsberg
Pandian, T.J., Sheela, S.G. and S. Kavumpurath, 1993. Endocrine sex reversal in fish-
Winge, O. and E. Ditlevsen, 1938. A lethal
using hydrostatic pressure. J. Aquac. Fish.
es: Masculinization evokes greater stress and
mortafity. Curr. Sci., in press.
Parsons, J.E. and G.H. Thorgaard, 1985.
Production of androgenic diploid rainbow
trout. J. Hered., 76:171-1 81.
Scott, A.G., Penman, D.J., Beardmore, J.A.
and D.O.F. Skibinski, 1989. The YY supermafe in Oreochromis niloticus (L) and its po-
tential in aquaculture. Aquaculture, 78:237251.
Varadaraj, K. and T.J. Pandian, 1989. First
repoft on production of supermale tilapia by
integrating
sex reversal with
gynogenetic
Ser. Physiol., 21: 1 -49.
gene in the Y chromosome
ol
Lebistes. C.R.
Trav. Lab. Carlsberg Ser. Physiol., 22',203216.
Yamamoto, T., 1955. Progeny of artificially
induced reversals of male genotype (XY) in
the medaka (Oryzias latipes) with special reference to YY male. Genetics,40:406-419.
Yamamoto, T., 1964. The problem of viability
of YY zygote in the medaka, Oryzias latipes.
Genetics,50:45-58.
Yamamoto, T., 1975. A YY male goldfish
from mating estrone induced XY female and
normaf male. J. Hered..66:2-4.