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Induction of tetraploidy in an ornamental fish koicarp
Cyprinus carpio L, using heat shock
ABSTRACT:

Koicarp is potentially an important cultured ornamental fish in freshwater.
Moreover there were reports existing on genetic manipulation of koicarp by
application of the heat shock. Hence the present study was made to contribute a
protocol for induction of tetraploidy by heat shock in the koicarp.Induction of
tetraploidy was attempted in Cyprinus carpio L, Koicarp by heat shock. Eggs from five
females and milt from five males ok Koicarp were pooled to ensure the required
quantity and quality of gametes for fertilization. After insemination the eggs were
divided into three batches each experiment based on the post fertilization viz., 25min,
27min and 30min after insemination. Batches of eggs held in plastic containers were
exposed to hot water at 38° C, 39° C, 40° C & 41° C for durations of 2min and four min.
One batch of the eggs without heat shock treatment was used as control. After
treatments, eggs were immediately transferred to incubation troughs. Tetraploidy
was ascertained by karyotyping as well as RBC nuclear micro measurements.Heat
shock of 41°C for four min, imparted to eggs for 20 min after fertilization induced a
maximum of 60± 2% tetraploidy and maximum hatchability of 10± 1.5%. A large
proportion of the heat shocked embryos displayed morphological abnormalities such
as short and curved tail, destroyed yolksac, deformed vertebral column and
malformed cephalic region. A maximum of 60± 2% tetraploids (4n = 156) were
obtained when the fertilized eggs (20 min old) were heat shocked at 41° C for four
min duration. The tetraploid red blood cells (RBCs) nucleus volume was 2.1 times
greater than those of the diploid RBC nucleus.Given that koicarp are such a useful
model for other areas of research, perhaps further studies on the induction of
tetraploidy in this species will lead to a better understanding of polyploidy induction
and the establishment of tetraploid lines of koicarp and other species as well.
013-019 | JRAS | 2012 | Vol 1 | No 1
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Journal of Research in
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Authors:
Ananth Kumar
1
and
Mohamed Abdul
Kadher Haniffa
2
.





Institution:
1. V.H.N.S.N
CollegeVirudhunagar
626001,Tamilnadu, India.

2. Centre for Aquaculture
Research and Extension
(CARE), St Xavier’s College
(Autonomous),
Palayamkotai-627002,
India.





Corresponding author:
Ananth Kumar.






Email:
annthneon@rocketmail.com,
labeoshark@gmail.com.

Phone No:
+91-4562-280154.

Fax :
+091-4562-281338.


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Dates:
Received: 16 Feb 2012 /Accepted: 22 Mar 2012 /Published: 13 Apr 2012
Article Citation:
Ananth Kumar and Mohamed Abdul Kadher Haniffa.
Induction of Tetraploidy in an Ornamental Fish Koicarp
Cyprinus carpio L, Using Heat Shock.
Journal of Research in Animal Sciences (2012) 1: 013-019
Journal of Research in Animal Sciences
An International Online Open Access
Publication group
Original Research


INTRODUCTION
Chromosome manipulation has become an
important tool to understand the interactions between
dissimilar or unequal genomic combinations and their
impact on survival, growth and reproduction in fish
(Pandian and Koteeswaran 1998). In the loach, Cobitis
biwae, (Kusunoki et al., 1994) produced the first bred
gynogen by heterologus activation of 2n eggs of
tetraploids and (Thorgaard et al., 1990) were the first to
produce a bred androgen activating the irradiated egg
with sperm from tetraploid salmon. Tetraploid rainbow
trout has been crossed with diploids to produce triploids
(Myers and Hershberger 1996). A good number of
publications are available on chromosome manipulation
in ornamental fishes and edible fishes mainly due to the
ease with which the gametes of oviparous fish can be
procured, fertilized in vitro and subject to induction or
after activation/fertilization. (Thrope et al., 1984;
Springate and Bromage, 1985; Tabata 1991; Pandian
1993; Cherfas et al., 1994, Horvath and Orban
1995;Jonson and Svavarsson 2000) and recent work with
other species (Gisbert et al., 2000; Ouellet et al., 2001;
Zaho et al., 2001; Pandian and Koteeswaran 1998 ,
Pandian et al., 1999 and Haniffa et al., 2004) made an
extensive effort to critically review various aspects of
chromosome manipulation. The underlying reason for
amenability of fish to different types of ploidy induction
is that ploidy induced fish (with exception to androgens
and paternal triploids) tolerate unequal genomic
contributions, so long as the genomic contribution of
female exceeds that of male (Pandian and Koteeswaran
1998). Studies on the occurrence of natural ployploids
have been reported in a number of fish species by
Pandian and Koteeswaran (1998 & 1999) in H.fossilis. In
male heterogametic species, induction of triploidy,
pentaploidy, hexaploidy and meiotic gynogenesis require
the retention of the second polar body but the production
of tetraploids, mitotic gynogens and androgens
necessasarily require the inhibition of the first cleavage
(Pandian and Koteeswaran 1998). A number of methods
have been developed to confirm ploidy in fishes as
monogenic and polyploid fish are not morphologically
distinguishable from diploids (Pandian and Koteeswaran,
1998). Among all methods, karyotyping is most widely
used to confirm ploidy. The presence of a marker
chromosome, in H.fossilis (Haniffa et al., 2004) and in
Oreochromis mossambicaus (Varadarj and Pandian
1988) can be used in the rapid identification at various
levels. Measurements of cell and nucleus size/ volume of
erythrocytes are other methods used to confirm triploidy
(Pandian and Koteeswaran 1998). Most of the authors
have chosen the combination of karyotyping and
measurement of erythrocyte to confirm ploidy. Another
potentially powerful technique for ploidy assessment is
isozyme variation and it has been effectively used to
separate gynogens or androgens or triploids from
diploids and mitotic gynogens in C.gariepinus (Na-
Nakorn et al., 2004).
Varadaraj (1993) alone succeeded in producing
live and gynogenetic, haploid Oreochromis
mossambicus, grass carp Ctenopharyngodon idella
(Cassani and Caton 1985)and, Nile tilapia Oreochomis
niloticus (Don and Avtalion 1988). Koicarp is potentially
an important cultured ornamental fish in freshwater.
Moreover, there are some reports that exists on the
genetic manipulation of koicarp by application of heat
shock. Hence the present study was made to contribute a
protocol for the induction of tetraploidy by heat shock in
the koicarp.

MATERIALS AND METHODS
Collection of eggs
Induction of tetraploidy in koicarp by heat shock
was attempted. Eggs from five females and milt from
five males of Koicarp were pooled to ensure the required
quantity and quality of gametes for fertilization. After
insemination the eggs were divided into three batches in
each experiment based on the post fertilization viz., 25,
014 Journal of Research in Animal Sciences (2012) 1: 013-019
Kumar and Haniffa, 2012
27 and 30min after insemination.
Treatment by using heatshocks
Batches of eggs held in plastic containers were
exposed to hot water at 38, 39, 40 & 41
0
C for the
durations of 2 or 4 min at each of the tested temperature.
One batch of the eggs without heat shock treatment was
used as control. After the treatments, eggs were
immediately transferred to incubation troughs. Dead eggs
were removed and the survivors were counted at
hatching.
Karyotyping
Chromosome preparation of the hybrids and
male parent (Koicarp) and female parent (goldfish) were
made following Sridhar and Haniffa 1999. The selected
fishes were kept alive in water containing 0.75%
colchine for six hours. The fishes were sacrified and their
gills, kidney and fins were dissected out. The tissues
were minced into small pieces (1 mm) and placed in
0.8% KCL solution (Hypotonic treatment) for 30
minutes. The tissues were individually fixed in methanol:
acetic acid (3:1) for 30 min with three changes of 10
minutes each. Tissues were then stored in the fresh
fixative in a refrigerator until further use.For slide
preparation the fixative was replaced by a few drops 50%
glacial acetic acid and agitated gently using a Pasteur
pipette.The tissue suspension (in acetic acid) was
expelled on the slides, heated to about 55
0
C on a slide
warmer.About 4 or 5 drops were expelled to each slide
and the suspension was quickly drawn back into the
Pasteur pipette. The slides were allowed to air dry. They
were stained in 5% geimsa stain made up in 0.01M
phosphate buffer (pH 6.8) for about 20 min.The slides
were rinsed in distilled water and air- dried. They were
observed for chromosome spreads under a microscope
(NikoE - 400). Tetraploidy was ascertained by
karyotyping (Haniffa et al., 2004) as well as RBC
nuclear micro measurements (Pandian and Koteeswaran
1998). The data were analyzed by Standard deviation
and means using Tukey’s multiple range test (Zar 2000)
to determine significant differences. The statistical
significance was calculated at [P<0.05%].

RESULTS
The percentage of tetraploids, diploids and
deformed fry resulting from heat shock experiments were
calculated. Heat shock at 41
0
C for 4 min, imparted to
eggs 30 min after fertilization induced a maximum of 60
± 2 % tetraploidy and maximum hatchability 58± 1.5 %
(Table 1). Among the treated eggs, majority of them
died before hatching or immediately after hatching. A
large proportion of the heat shocked embryos displayed
morphological abnormalities such as short and curved
tail; destroyed yolksac; deformed vertebral column and
malformed cephalic region (Fig. 1-4). Heat shock below
40
0
C proved to be 60 % survival and about 18± 3.5%
(Table 3) of the induced tetraploids were deformed when
Journal of Research in Animal Sciences (2012) 1: 013-019 015
Kumar and Haniffa, 2012
Table 1. Effect of heat shock (38
0
C )on survival at hatching and tetraploid induction in Koicarp Cyprinus
carpio. Each value represents the average of three repetitions and ± indicated the standard deviation.
Time after
fertilization (Min)
Shock duration
(min)
No. of eggs Hatching
(%)
Survival
(%)
Tetraploid
(%)
Deformed
(%)
5
5
10
10
15
15
20
20
2
4
2
4
2
4
2
4
100
100
100
100
100
100
100
100
72.3 ± 2.5
64.3 ± 4
55.6 ± 4
63.3 ± 4.16
56 ± 4
70.3 ± 1.5
64 ± 1.4
65.0 ± 3

54.3 ± 4
40.0 ± 5
57.6 ± 2.5
59.3 ± 5.1
41.0 ± 3.6
70.0 ± 5
70.0 ± 5
62.3 ± 2.5
0
0
0
0
0
0
0
0

0
0
1
1
0
0
0
0
Deformed fry include diploid and haploid


compared to only 4 ± 2 % and 2% deformed fry at 39
0
C
(Table 2) and in control, only deformed was observed
(Table 4). Among the tetraploid induced individuals
none survived to feeding stage (4 days after hatching).
The tetraploidy was confirmed by chromosome counts
and erythrocyte nuclear volumes. The metaphase spreads
of diploid control (2n = 78) and tetraploid (4n = 156) are
shown in (Fig. 5-6). The nuclear volume of diploid
RBCs was 8 ± 2 μm
3
and that of tetraploid was 19 ± 2.5
μm
3
(Fig. 7-8)

.

DISCUSSION
The results of the present study showed that
tetraploidy could successfully be induced in koicarp by
heat shocking 4 min and 30 min old eggs (post
fertilization) respectively at 41
0
C for 4 min duration.
Previous studies have shown that in most tropical fishes
the extrusion of second polar body can be inhibited by
heat shocking 2-4 min old eggs at 40 to 42
0
C for 2-5 min
duration (Varadaraj and Pandian1988 and Haniffa et al.,
2004). Till date it has been possible to produce live
tetraploids in about few species. A survey of the relevant
literature shows that the optima protocol of tetraploidy in
fishes varied from species to species. Thus, in the present
study 25-30 min old embryos were used for heat
shocking. Tetraploid embryos of koicarp were obtained
when heat shock was applied 30 min after fertilization.
The doubling of chromosome was due to suppression of
first cleavage. 4n embryos successfully produced in
O.niloticus (Myers, 1996) and O.mossambicus (Pandian
and Varadaraj, 1987) where as H.fossilis (Haniffa et al.,
2004) failed to survive in first feeding stage. In this
present study also 4n koicarp hatchlings failed to survive
till first feeding. Low yields of 4n at other temperatures
016 Journal of Research in Animal Sciences (2012) 1: 013-019
Kumar and Haniffa, 2012
Table 2. Effect of heat shock (39
0
c )on survival at hatching and tetraploid induction in koicarp cyprinus
carpio. Each value represents the average of three repetitions and ± indicated the standard deviation.
Time after
fertilization (Min)
Shock duration
(min)
No. of eggs Hatching
(%)
Survival
(%)
Tetraploid
(%)
Deformed
(%)
5
5
10
10
15
15
20
20
2
4
2
4
2
4
2
4
100
100
100
100
100
100
100
100
68 ± 2.6
61 ± 3.6
58 ± 2
54 ± 2
58 ± 2
54 ± 2
50 ± 5
53 ± 2

44 ± 4
42 ± 2
34 ± 4
33 ± 4
36 ± 1
34 ± 2
36 ± 4
36 ± 1.5
0
0
0
0
0
0
4 ± 2
0
0
0
0
0
0
0
2
0

Deformed fry include diploid and haploid
Table 3. Effect of heat shock (40
0
C )on survival at hatching and tetraploid induction in Koicarp Cyprinus
carpio. Each value represents the average of three repetitions and ± indicated the standard deviation.
Time after
fertilization (Min)
Shock duration
(min)
No. of eggs
Hatching
(%)
Survival
(%)
Tetraploid
(%)
Deformed
(%)
5
5
10
10
15
15
20
20
2
4
2
4
2
4
2
4
100
100
100
100
100
100
100
100
62 ± 2.5
60 ± 2
56 ± 1.5
53 ± 1.5
52 ± 3
50 ± 1.5
50 ± 1
46 ± 2
47 ± 2.5
55 ± 3
50 ± 1.5
49 ± 2
46 ± 1.5.
47 ± 2
50 ± 1.5
50 ± 4.7

0
0
0
0
0
11 ±1.20
0
18 ± 3.5
3
0
0
0
2
5
0
8
Deformed fry include diploid and haploid
(38, 39 & 40
0
C) at embryo ages higher or lower than 30
min may be due to the inability in suppression of 1
st

cleavage of the zygote. The exact time of shock applied
should correspond to karyokinesis or cytokinesis. In
salmonids species treatment time for inhibition of
karyokinesis are resulted in better survival Pandian and
Koteeswaran 1998. The developmental abnormalities
observed in the study appeared to be caused by the heat
shocks and not the presence of extra sets of chromosome
in the tetraploids. This assumption is based on the fact
that some normal appearing fish were tetraploid and
some abnormal fish were diploid. Studies on
chromosome set manipulation in other fishes have found
abnormal appearances that the fish to be a diploid
(Haniffa et al., 2004) adding support to the hypothesis
that abnormalities result from the shocks and not from
the extra chromosome sets.
The tetraploid RBCs nuclear volume was on an
average 1.5 and 2.1 times greater than that of the diploid
RBCs nuclear volume respectively. Similar results in
RBCs nuclear volume were reported by Haniffa et al.,
(2004) in triploid and tetraploid H.fossilis. The
chromosome number for diploid (2n = 78) and tetraploid
(4n = 156) koicarp obtained in the present study
correspond with the observations of Pandian and
Koteeswaran 1999 in natural polyploids. According to
Haniffa et al., (2004) the chromosome number of diploid
and tetraploidy H.fossilis were as 2n = 58 and 4n = 116.
CONCLUSION
Given that koicarp are such a useful model for
other areas of research, perhaps further studies on the
induction of tetraploidy in this species will lead to a
better understanding of tetraploidy induction and the
establishment of tetraploid lines of koicarp and other
species as well.
Journal of Research in Animal Sciences (2012) 1: 013-019 017
Kumar and Haniffa, 2012
Table 4. Effect of heat shock (41
0
C ) on survival at hatching and tetraploid induction in Koicarp Cyprinus
carpio. Each value represents the average of three repetitions and ± indicated the standard deviation.
Time after
fertilization (Min)
Shock duration
(min)
No. of eggs
Hatching
(%)
Survival
(%)
Tetraploid
(%)
Deformed
(%)
5
5
10
10
15
15
20
20
2
4
2
4
2
4
2
4
100
100
100
100
100
100
100
100
40 ± 0.5
58 ± 1.5
24 ± 4
19 ± 1
0
0
0
10 ±1.2
24 ± 4
43 ± 3
15 ± 3
10 ± 1.5
0
0
0
8 ± 3
13 ± 1.5
30 ± 1.5
6 ± 4.1
0
0
0
0
60 ± 2
12
10
0
0
0
0
0
15
Control 100 60 ± 2 70 0 0
Deformed fry include diploid and haploid


ACKNOWLEDGMENTS
We sincerely thank Rev. Dr A. Alphonse
Manickam S.J., Principal, St Xavier’s College,
Palayamkottai, for providing necessary facilities.

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