induction of tetraploidy in an ornamental fish koicarp cyprinus carpio l, using heat shock

Jou

rn

al of R

esearch

in

A

nim

al Scien

ces

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

© Ficus Publishers.

This Open Access article is governed by the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which gives permission for unrestricted use, non-commercial, distribution, and reproduction in all medium, provided the original work is properly cited.

Submit Your Manuscript

www.ficuspublishers.com http://ficuspublishers.com/

Journal of Research in

Animal Sciences An International Open Access Online

Research Journal

Authors:

Ananth Kumar1 and

Mohamed Abdul

Kadher Haniffa2.

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:

[email protected],

[email protected].

Phone No:

+91-4562-280154.

Fax :

+091-4562-281338.

Web Address: http://ficuspublishers.com/

documents/AS0006.pdf

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 & 410C 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 550C 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 410C 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

400C 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


Table 1. Effect of heat shock (380C )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 390C

(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 μm3 and that of tetraploid was 19 ± 2.5

μm3 (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 410C 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 420C 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



Table 2. Effect of heat shock (390c )on survival at hatching and tetraploid induction in koicarp cyprinus


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


Table 3. Effect of heat shock (400C )on survival at hatching and tetraploid induction in Koicarp Cyprinus


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


(38, 39 & 400C) at embryo ages higher or lower than 30

min may be due to the inability in suppression of 1st

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.



Table 4. Effect of heat shock (410C ) on survival at hatching and tetraploid induction in Koicarp Cyprinus


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


ACKNOWLEDGMENTS

We sincerely thank Rev. Dr A. Alphonse

Manickam S.J., Principal, St Xavier’s College,

Palayamkottai, for providing necessary facilities.

REFERENCES:

Cassani JR and caton WR. 1985. Induced triploidy in

grass carp, Ctenopharyngodon idella val. Aquaculture

46:3-44.

Cherfas NB, Peretz Y, Bendom N, Gomelsky B and

Hulata G. 1994. Induced Diploidgynogenesis and

polyploidy in the ornamental (koi) carp, Cyprinus carpio

L.4.Comparative-study on the effects of high-

temperature and low-temperature shocks.Theoretical and

Applied Genetics, 89:193-197.

Don J and Avtalion DR. 1988. Production of viable

tetraploid tilapias using the cold shock technique,

Bamidgeh 40:17-21.

Gisbert E, Williot P and Castello-Orvay F. 2000.

Influence of egg size on growth and survival of early

stages of Siberian sturgeon (Acipenser baeri) under small

scale hatcheryconditions. Aquaculture, 183:83-94.

Haniffa MA, Sridha S and Nagarjan M. 2004.

Introduction of triploidy and tetraploidy in stinging

catfish, Heteropneustes fossilis (Bloch) using heat

shock . Aquaculture Research, 35:937-942.

Horvath L and Orban L. 1995. Genome and gene

manipulation in the common carp, Aquaculture 129:157-

181.

Jónson B, Svavarsson E. 2000. Connection between

egg size and early mortality in arctic charr, Salvelinus

alpinus. Aquaculture, 187:315-317.

Kusunoki T, Arai K and Suzuki. 1994. Production of

viable gynogens without chromosome duplication in

Spinous loach, Cobitus biwae, Aquaculture 119:11-19

Myers JM and Hershberger WK. 1996. Artificial

spawning of tilapia eggs. Journal of world Aquaculture

society 22:77-82.

Ouellet P, Lambert Y and Bérubé I. 2001. Cod egg

characteristics and viability in relation to low

temperature and maternal nutritional condition. ICES

Journal of Marine Science 58:672-686.

Pandian TJ. 1993. Endocrine and chromosome

manipulation techniques for the production of all male

and all female population in food and ornamental fishes

Proc. Ind. Nat.Sci.Acad.59B:549-566.

Pandian TJ and Varadaraj K. 1987. Techniques to

regulate sex ratio and breeding in tilapia. Cur.Sci.,

56:337-343.

Pandian TJ and Koteeswaran R. 1998. Ploidy

induction and sex control in fish. Hydrobiologia 384:167

-243.

Pandian TJ and Koteeswaran R. 1999. Natural

occurrence of monoploids and polyploids in the Indian

catfish, Heteropneustes fossilis. Current Science 76:8,

1134 -1137.

Na-Nakorn U, rangsin W and Boon-ngam J. 2004.

Allotriploidy increases sterility in the hybrid between

Clarias macrocephalus and C.gariepinus. Aquaculture,

237:73-88.

Springate JRC and Bromage NR. 1985. Effects of egg

size on early growth and survival in rainbow trout

(Salmo gairdneri R). Aquaculture, 47:163-172.

Thorgaard Gh, Scheerer PD, Hershbergar Wk and

Myers JM. 1990. Andrigenetic rainbow trout produced

using sperm from tetraploid males show improved

survival, Aquaculture 85:215-221.

Thrope JE, Miles MS and Keay DS. 1984.

Developmental rate, fecundity and egg size in Atlantic



salmon, Salmo salar L. Aquaculture, 43:313-322.

Tabata K. 1991. Application of chromosomal

manipulation in aquaculture of Hirame, Paralichthys

Olivaceus. Bull. Kyogo Pref. Fish. Exp. 28-134.

Varadaraj K. 1993. Endocrine and genetic studies on

sex regulation in tilapia. Ph.D. thesis, Madurai Kamaraj

University, Madurai India 55.

Zaho YC and Brown JA. 2001. Impacts of egg size and

larval size on survival and growth of Atlantic cod under

different feeding conditions. Journal of Fish Biology, 59:

569-581.

Zar JH. 2000. Biostatistical analysis (2nd Eds) Prentice -

Hall International, Inc., Englewood Cliffs, New Jersery.



Submit your articles online at Ficuspublishers.com

Advantages

Easy online submission Complete Peer review Affordable Charges Quick processing Extensive indexing Open Access and Quick spreading You retains your copyright

[email protected]

www.ficuspublishers.com/submit1.aspx.

induction of tetraploidy in an ornamental fish koicarp cyprinus carpio l, using heat shock

Documents