Induced triploidy in the common carp (Cyprinus carpio L.): a

Aquur.
Li~irigResour., 1991, 4, 139-145
Induced triploidy in the common carp (Cyprinuscarpio L.):
a cornparison of two methods
Otomar Lirihart, Martin Flajghans and Pave1 KvüsniCka
K e ~ r a r c hItutitute qf f i s h Ci111i~reand /~drohiolo,v,
Ue/~c~rirnerii
of 1.lth G'eneiic~and Brerdin,q,
:j!!rj 2.5 l'ocl6irtiry, (,>et/lu,looakia.
Rcccivccl.January 211,
r!y)i;
acrrj>tc[lXIay fi,1991.
Linhart O., M. FlajShans, P. Kvasnitka. Ayuol. I,icing Resour., 1991, 4, 139-145.
Abstract
Cold shocks and hydrostatic pressure shocks, rcspcctivcly, wcrc applied Tor the induction of hoth
triploidy and tetraploidy in thc common carp. Cold shocks of 0-2"C, lasting 40 minutcs and starting
2-5 minutes after the activation of gametcs and hydrostatic prcssure shocks with 1-3-5 minute cxposurcs
at 49.03-56.88 MPa (i. e. 7 116-8225 psi - pounds per squarc inch), starting 5 minutes after the
activation of gametes gavc thc bcst rcsults. The tetraploidy induction by cold shocks or hydrostatic
prcssure shocks resulted either in negligiblc (1.54%), or in zero yields, respectively. The applicability
of particular typcs of ploidy manipulation and the standardization of shock initiation were discussed.
Keywords :Carp, triploidy and tctraploidy induction, cold shocks, hydrostatic pressure shocks.
Triploïdie induite chez Iu carpe commune (Cyprinus carpio L.) :compurai~sonde deux méthodes.
Résumé
Des chocs thcrmiqucs froids et des chocs de pression hydrostatique sont appliquts respectivement
afin d'obtenir des carpes triploïdes et tétraploïdes. Des chocs donnant les meilleurs résultats sont: Ics
chocs thermiqucs de 0-2"C, d'une durée de 40 minutes et commençant 2 à 5 minutes après l'activation
des gamètes; des chocs dc prcssion hydrostatique, d'une durée de 1, 3 et 5 minutcs dc 49,03 à
56,88 MPa (équivalents à 7 116 et 8225 psi) débutant 5 minutes après l'activation des gamètes.
L'induction de la létraploïdie au moyen de chocs therrniquc froids est négligeable (1,54 %) ou nulle
au moyen de chocs dc prcssion hydrostatique. Les conditions d'application de manipulations en vue
d'obtenir des types particuliers de polyploïdie sont discutées ainsi que la standardisation de l'initiation
des chocs.
Mots-clés : Carpe, triploïdie, tétraploïdie, induction génétique, chocs thermiques, pression hydrostatique.
INTRODUCTION
Two physical methods, viz. temperature shocks and
hydrostatic pressure shocks, have becn successfully
applied by various authors for the retention of the 2nd
polar body during meiosis and for the suppression of
Aquat. Living Resour. 91/03 139 07 $ 2.7010 IFREMER-Gauthier-Villars
chromosome disjunction before the 1st and/or 2nd
mi tosis. Thorgaard (1983, 1986), Chourrout (1987),
and Benfey (1987)7 Benfey (1989) and
others reviewed the respective experiments and discussed the possibilities of application of these manipulations in fish culture. It was previously believed, that
140
hcat shocks are inore eflicient for induction of polyploidy in cold-watcr fi\hcs than cold shocks and vice
versa. IIowever. the expcrimcnts of Strciringcr et rrl.
(1981), Chourrout and Itskovich (1983). Hidwcll r t ul.
(1985) and Hollcbccq ct (11. (1988) have shown, that
heat shocks can bc efficient in wnrni-water fishes also.
The optimi~ation of ploidy inanipulation parametcrs (i. p. tcmpcraturc o r hydrostatic prcssurc uscd,
duration of the shock and the timing of the initiation
o f the shock aftcr gamete activation) in both types of
physical shock is the kcy point of the whole procedure
because the egg sensitivity changes rapidly during
carly embryonic dcvelopment (Cherfas et al., 1990).
In various experiincnts with ploidy manipulations the
fcrtili/cd cggs wcrc kept at various ternperatures prior
to shock which itsclf has bccn applied in different
time intervals after fertiliration. In ordcr to 0ptimii.c
the shock initiation, Gomelsky et cd. (198'9) and
Cherfas rt 01. (1990) have tested a dimensionless unit
of embryonic age r, [the duration of one initotic cycle
during synchronous cell division (Ilctlaff and 1)ctlaft;
1961; lgnatyeva, 1974)l related to the watcr tcmpcraturc only. Artificial triploidy in common carp or koi
carp has bccn succcssfully induced in experiments of
Ojima and Makino (1978). Gervai et ul. (1980), Ueno
(1984), Tanig~ichirt rd. (1986), Wu et al. (1986), Hollebecq ct (11. (19881, Linhart cf cd. (1989) and Cherfas
ct al. (1990). Most of these authors used cold shocks,
exccpt Hollcbccq rt (11. (1988) and Rekoubratsky
ct LI]. (1980), who uscd the heat shock. Artificial tetraploidy was successfully induccd by Rekoubratsky
ct (11. (1989) by heat shock only. To the bcst of our
knowlcdge, the application of hydrostatic prcssurc
shock has not bcen used for ploidy manipulation of
common carp or othcr closcly related species except
by Cassani and Caton (1986) for grass carp.
We report in this papcr the rcsults of Our experiinents with artificial induction of triploidy and tctraploidy in common carp by both cold shocks and
hydrostatic pressure shocks.
MATERIAL ANI) ME'I'HODS
Al1 experiments wcrc carried out at the Experimenta1 Station of Research Institute of I:ish Culture and
Hydrobiology, (RIFCH) a t Vodiiany in 1987 and
1989. Hrood carps, originating from strains kept in
RIFCH at Vodiiany, were hcld in warmed water prior
to artificial stripping. 7'hc duration of this period
varied according to the dcgrcc of ripcncss of the
oocytes in females used in single experiments. Fish
wcre stripped by the conventional dry method. Gametes werc chcckcd macro- and microscopically in order
to cxcludc spcrm with poor motility, sperm contaminated with urine or faeccs or ovcrripcd oocytes. Heterosperin, after mixing from individual males, was
maintained in glass tubes in refrigerator a t 4-5°C prior
to the cxperiments. Oocytes mixed from individual
0. Linhart et al.
fcmalcs wcre maintained in plastic bowls covercd with
a wet cloth in ordcr to prcvcnt drying. Gametes
were also collected for estimation of the aniount of
spermatozoa in I ml of sperm and the number of
oocytes in 1 g.
Generally, the schedule or al1 cxpcrimcnts was as
follows: oocyte batches (5-10 g) were weighed in glass
di\hcs, spcrm was addcd, mixed and activatcd with
20 ml of dcgumming solution consisting of milk:
water ( 1 :40). After 2 minutes a morc concentrated
(1 : 30) milk solution was addcd. The batchcs of cggs
wcre incubated in Kannengieter incubation jars (2 1
volume) in non-recycled warmed water at an average
temperature of 20°C. Each jar was placed i i i one
aquarium, irito which the Iarvac were transfercd aftcr
hatching. Just before starting exogenous feeding, the
total nuinber of larvae was checked and a representative sample was takcn to dctcrminc ploidy level by
quantification of Ag-stained nucleoli in interphase
cclls andior Ag-stained nucleolar organizer regions
(NOKs) in the inetaphasc chromosomes. For this
detcrmination, the inethod of Phillips et al. (1986)
has been utili/cd but modificd according to Fl2ijShans et (11. (in press u ) . Briclly, the wholc larvac wcre
smeared on ethanol-cleaned microscopic slides in one
drop of 60% acetic acid, fixed with methanol, dried
and Ag-staincd according to Howell and Black (1980),
modified by Gold (1984) and Rab and Roth (1988).
Chromosome preparations were made as dcscribcd
by Baksi and Means (1988).
The experimental batches were treated with crushcd
ice dispersed in rnilk solution (1 :20) with the tcmpcraturc 0-2°C. The shocks were initiated from O to
65 minutes, rcspcctively, after gamete activation (see
tuh1e.r 1 and 2). the cxposurc lastcd for 40 minutes,
(Linhart r t ul., 1987). The average temperature prior
to the exposure was 24°C. '
A hydrostatic pressure device dcvclopped for
RIFCH at Vodfiany by Moravian Chcmical Entcrprise at Ostrava (courtesy Dr. Theobald Olschak;
device undcr patent control) has been used. In this
device pressurized nitrogen is uscd to provide the
hydrostatic pressure, so that the required pressure
lcvcl can be reached in less than two seconds.
The tempcraturc of watcr and the milk solution in
the pressure chamber rangcd from 19 to 21°C. Egg
batches were treated in the pressure chamber in bags
made from plastic mesh. The eggs were incubatcd
aftcr shocking a t an average temperature of 20°C.
Threc expcrimcntal scrics wcre realized, in each series
one shock parameter (starting time of shock after
activation, pressure level, exposure time, rcspectively)
was tested, while the remaining two wcrc kcpt constant in order to optimize the parameters a t the tempcraturc used.
Percentuge of hutched ciuhle polyploid frjl:
percentage of hatched viable fry x pcrccntagc o f
po]yploidç from the analy& samplcs.
As incxact shocks can significantly affect the survival of incubated eggs, the results reported in this
paper were calculated from the numbcr of living cggs
at morula stage in each experiniental batch as follows.
Licing eggs ut moruiu .ytugcJ:
number of eggs in cxperimental batch x average
percentage of living eggs at morula stage from 5
analyzcd random samples.
Percentugc of hutch~.dcic~hlefry:
number of hatched viable fry/numbcr o f living eggs
at morula stage.
REÇULTÇ
Colcl shocks
The results of cxpcrimcnts for the induction of
triploidy arc givcn in tühlc>1 and in figures 1 and 2.
Initiation of thc shock [min]
Initiation of the shock [min]
Figrre 2. - Cold shocks: pcrccntagc of viable triploid fry (3 I I ) for
replicated groups (1 and II scrics).
Figure 1. - Cold shocks: percentagc of viahlc fry for two replicated
groups (1 and II series).
Tslk 1.
-
two
Cold shocks.
~
~
Initiation of the shock aftrr garnete activation (min)
Living eggs
at morula stage (%)
Viable fry (%) +
*
Viable 3n fry (%) **
Viable 4n fry (%) *
Viablc 4n fry (%) **
Viable 3n fry (%)
O
1. 94.0
11.
1. 93.56
II.
1.
15.26
II.
1. 15.79
II.
1.
II.
1.
11.
2
88.0
86.0
93.56
97.68
73.87
61.69
78.95
63.16
5
81.0
86.0
93.42
94.96
74.74
75.97
W.00
EO.OO
15
20
25
30
43
1.0
71.0 80.0 55.0
82.0 61.0
1.0
83.0 82.0 66.0
89.0 85.0
18.98 7.68 33.32 3.44 0.32 2.42
6.45 18.67 0.34
1.15 0.29 12.12
2.1 1
1.38
0.06
11.1 1
21.43
5.00
Nurnbers 1, II are numbcrs of cxpcrimental series.
C is the abbreviation of control group (untrcatcd group).
percentage was calculated from the percentage of living eggs at morula stage.
pcrccntagc was calculated from the absolute number of viable fry.
*
**
35
45
2.0
4.0
8.64
7.18
50
..
55
60
16.0
5.0 55.0
8.0
28.0 17.0
3.00 1.79 0.38
2.22 1.45 3.43
.
65
-
-
93.0
1.85
-
95.5
0.13
0.22
1.54
7.14
15.00 45.00
C. -
6.0
-
The bcst rcsults were obtained after shocks 2 and 5
minutes after activation of gainetes i.e. 93.56 and
97.68% of hatched viable fry with 73.87 and 61.69'%1
of hatchcd viablc triploid fry, rcspcctively for two
rcplicatcd groups shocked after 2 minutes; and 93.42
and 94.96% of hatched viable fry with 74.74 and
75.97% of hatched viable triploid fry, rcspcctivcly, for
thc shock aftcr 5 minutes (tuhlr l,,/ïg. 2). If expressed
in r,, ~ i n i t ,it cquals 0.1 and 0.26 at the incubation
hater temperature of 24°C for 2 and 5 minutes
shocks, respectively.
7'hc percentage success for the induction of tetraploidy proved to be negligible (e.g. 1.54, 0.22 and
0.13% of hatched viable tetraploid fry from cold
shocks after 55,60 and 65 minutes of incubation,
respectively) and thcy werc rcachcd in one cxpcrimcnt
only with n o successfull replication (ruhl~>
1, ,Jg. 3).
However, these tetraploids were safely produced by
cold shock, according to the fact, that nothing but
diploids wei-e found in thc respective control groups.
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Initiation of the shock [min]
('olcl shocks: pcrcciitagc of viablc Iciraploi(i fry (411).
Hj~~Iro.stcrtic
pr-r.s.surc s h o c k . ~
best results (63.49% and 52.29% of hatchcti viablc
triploid fry) were obtained using the shortest exposure
T'hc rcsults of experimcnts for the induction of
triploidy are given in luhle 2, in /ïguvc.c 4 and 5. The
TaZe 2.
Ilqdro~tnticp r c ~ s u r csliocks, 5 min aficr gûmctc activation.
...-
Expcrimcnt
No.
1
2
3
4
5
6
7
8
9
10
II
12
13
14
15
I0
17
18
IO
20
21
22
23
24
25
2 (1
C1
C2
Pressure
(MPa)
Expobure
(miii)
-- .
-- . .
50.00
10.79
20.10
30.89
39.23
49.03
51.98
52.96
56.88
40.03
49.03
48.25
47.37
45.60
44.13
43.15
55.90
9.8 1
20.59
30.40
39.72
50.99
5 1 .O8
53.94
53.94
55.00
.- . ..
Living eggs at
morula stage (%)
I
1
I
1
1
2
3
4
5
h
7
8
10
20
20
20
20
20
20
20
3O
40
-
O
.
..
.
22.0
10.5
7.5
8 .O
13.5
5.5
6.5
24.0
29.0
19.5
13.5
13.5
15.5
9.5
5.0
6.0
0.03
1
1
1
... ..
4.5
8 .O
3.5
2.5
O
O
O
O
O
39.0
24.6
-- .
Viable
fry (%)
*
82.35
19.52
2 1.22
18.75
56.60
1 .O9
18.46
76.74
61.01
76.28
80.19
49.38
50.65
28.16
17.17
54.03
O
O
82.18
14.97
O
O
O
O
O
76.3
94.08
.-...
.
.- . . . .
Viable 3n
fry (%) t t
20.59
O
O
O
1 1.79
O
7.69
19.19
52.29
30.5 1
03.49
39.50
42.21
23.26
13.44
36.02
O
O
60.07
O
O
O
25.0
O
O
O
O
O
O
O
- .- - .--
Viable 3n
fry (o/;,) *
O
20.83
O
41.67
25.0
85.7
40.0
79.17
80.0
83.33
82.0
78.26
66.67
O
O
73.10
O
O
O
O
O
O
O
-
-
-
-
P
.
-
- -
thc abhrekiation of control group (untreated group, 2 replications).
+ perccntapc w35 calculatc<l froiii the pcrccntagc o f living cgps al morula stage.
t r perceritagc \\a\ calculated Ii.orn the ab5olutc numbcr of viablc Sry.
(' iç
Acliiat.
Living Resour.
Induccd triploidy in the cornrnon carp
143
pressure levels did not rcsult in significant numbers
of hatched viable fry or in triploids at all, cxcept for
60.07% triploids from the shock at 20.59 MPa for 20
minutes exposure and for 14.97% viable fry after 20
minutcs exposure to a shock of 30.40 MPa. Howevcr,
thcse data were not repcatable.
Hydrostatic pressure shocks of 49.03 MPa, starting
at 40, 45 and 50 minutes after gainetc activation, i. e.
at 1.43; 1.61 and 1.79 T, and lasling 1 minute were
not succcssful for the induction o î tetraploidy in any
of Our cxperiments.
DISCUSSION
Exposure time [min]
Figure 4. - Hydrostatic pressure shocks related to cxposurc time:
percentage of viable fry.
Exposure time [min]
Figure 5. - Hydrostatic pressure shocks: pcrcentage of viablc
triploid fry (3 n).
times (1-3-5 minutes) for the highest hydrostatic pressure levels (49.03-56.88 MPa, i. e. 7 1 16-8255 psipounds per square inch) after 5 minutes incubation,
i. e. in 0.17 r, unit at an incubation water temperature
of 20°C. Shocks starting after 2, 10, 15, 20, 25 and
30 minutes incubation did not result in any viable fry
at all. Shocks with long exposure times at lower
Vol. 4, no 3 - 1991
The fertilization rates in each expcriment were
theoretically at the same level as in control groups
and they wcrc reduced to the observed level by the
shock trcatments (tubles 1 and 2). We tricd to
transform the results of the experiments to a mode,
which would reflect the correlation betwecn the fertilization rate and thc influence of the shock treatment
itself i. e. the survival, expressed in the percentage of
hatched viable fry and the "yield", expressed as thc
percentage of hatched viable polyploid fry. Thereforc,
the pcrcentage of hatched viable polyploid fry was
calculated from the number of living eggs at the
morula stage and not directly from the number of
hatched viablc fry; the latter mode of calculation was
taken only as a subsidiary item (tuhlc..~1 and 2).
Cold shocks
If the rcsults of previously published cold shock
experimcnts are transformed to T, parameter, the data
for the retention of the second polar body range from
0.03 (Cherfas et al., 1990) to 0.45 (Taniguchi et ul.,
1986), with the pcak in 0.03-0.32 interval (Gervai
et al., 1980; Ueno, 1984; Taniguchi et ul., 1986; Lin.hart et al., 1989; Cherfas et al., 1990). The data of
our experimcnts (0.1 and 0.26 r, for shocks at 2 and
5 minutes, respectively) thus correspond with them.
The best data for suppression of the first mitosis
were reported by Rekoubratsky et al. (1989) at 1.7
T,. In our experiments hatched viable tetraploid fry
were produced at a very low percentage (up to 1.54%)
and without successfull replication, if expressed in r,
unit, it ranged from 2.89 to 3.42. We suppose, that
this could be a result of suppression of the second
mitosis, while shocks applied before the first mitosis
did not induce tetraploidy at all.
Hydrostutic pressure shocks
As no data on the application of hydrostatic pressure shocks for induction of polyploidy in carp are
available, we have arranged Our cxperiments according to Cassani and Caton (1986). It is evident from
figures 4 and 5, that triploidy inducing shocks with
the shortest exposure times for the highest pressure
levels applied shortly after the gamete activation are
144
m o r e efficient than longer shocks with lower pressure.
T h e observation o f 60.07'% hatched viable triploids
after 20 minutes shock o f 20.59 M P a m a y b e a result
of a mistakc in a process o f fish handling (e.g.
replacement o f aquaria, etc.).
Hydrostatic pressure shocks were not successful for
the induction o f tetraploidy in c a r p in a n y o f o u r
cxpcrimcnts. I n contrast, very similar cxpcriments o n
tench (Ï'it~(.utirzcu L., Cyprinidae) resultcd in signilic a n t numbers of tctraploids ( u p t o 50%) b y t h e suprcssion of t h e first mitosis (I:laj<hans cJt al., in press h).
Hydrostatic pressure shocks d o not sccm t o be t h e
most efficient way for induction o f triploidy in carp,
n o t only d u e t o lowcr yiclds rcachcd. but also d u e t o
difficulties with handling during t h e treatment process. In prcvious experiments, where the pressure
O. Linhart et ul.
chamber was fillcd with water. al1 eggs of cach treatcd
batch wcrc stuck together a n d it was n o t easy t o
relcase thcm, thcrcfore we filled t h e charnber with
degumming milk: water (1 :20) solution t o reducc t h e
stickncss o f t h e eggs, which is the strongest in t h e
first minutes after gametc activation, when contcmporarily t h c triploidy-inducing trcatments start. This
arrangement proved t o b e partially succe~çful.
W e believc that teinperature shocks a r c m o r e cffèctivc thaii hydrostatic pressure shocks i n carp,
especially t h e heat shocks, with shock exposures causing lowcr niortality (Hollebecq ct (il., 1988; (iomelsky, pers. comn-i.). F o r t h e standardimtion a n d t h e
cornparison o f d a t a o n shock initiation, t h e application o f t h e s,, unit (Detlaff a n d Dctlaff, 1961; Ignatyeva, 1974; Gomelsky r t (il., 1989 Cherfris cl al.,
1990) seems t o b e essential.
Acknowledgements
Our thanks arc duc to Dr. Daniel Chourrout, INRA, Jo~iy-cn-.losas, I:rarice, Ilr. Boris 1. Goinclsky, Fishcric\ Ucscarch
Institutc (VNIIPRCH), Rybnoye, USSR and Dr. Pctr KBh, IAI'G, Dcpartmcnt of Gcnctics, C'SAS, for kindly rcvic~ving
tlic inanuscript and Mrs. Danicla Odlcvikovi for tcchnical assistance.
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