Bencsik I., et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
Triploidy determination in rainbow trout (Oncorhynchus
mykiss) based on erythrocytes dimensions
Ioan Bencsik1, Nicolae Pacala1, Gabi Dumitrescu1, Dorel Dronca1, Jana Stanculet2, Liliana
Petculescu-Ciochina1
1
Banat's University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and
Biotechnologies, 300645, Calea Aradului Street, No. 119, Timisoara, Romania
2
SC Detergenti SA, 300571, Calea Stan Vidrighin,No. 5,Timisoara, Romania
Abstract
Triploidy induction at fish is characterized by modification of normal diploid chromosome set (2n) into triploid set
(3n). Experiments were carried out on biological material from rainbow trout (Oncorhynchus mikiss) during the
natural breeding season. Polyploidy was induced by exposing the eggs to heat shock. Blood smear was used as a
technical method, to determine diploid and triploid status. Staining of blood smear was performed by Pappenhein
method. The erythrocytes area and perimeter measurements done comparatively on triploid and diploid individuals
may represent an indicator to determine the ploidy level of individuals. Erythrocytes area (µm²) for triploid
individuals is 1.76 times higher than at diploid individuals, and perimeter (µm²) 1.31 for triploid individuals is times
higher than in diploid individuals.
Keywords: erythrocytes, heat shock, rainbow trout, tetrapoidy
1. Introduction
in polyploids applies to all tissues and cells of the
body. Thus by measuring and comparing the cell
size we can distinguish diploid individuals and
triploid.
Polyploidy determination can be achieved by
karyotyping (visualization and counting of
chromosomes), flow cytometry, cell size
measurement by Coulter Counter Channellizer or
blood smear, silver staining of nucleolar
organizing regions (RON), isozymes analysis
using markers [10, 11]. Karyotyping is the most
accurate method, but the technique is slow and
laborious [12,13]. Flow cytometry is quick
method but the equipments are very expensive.
Coulter Counter analysis is rapid, equipment is
cheaper. Measurement of the size of blood cells in
the smear method is quite accurate [10,11].
Analysis RON is the cheapest and easier to
perform than karyotyping, it can be applied
without sacrificing the young fish. The principle
of the method is based on determining the number
RON (1 RON per haploid genome) [14,15].
Aim of the study was to determine triploid
individuals obtained after thermal shock
application on fertilized rainbow trout eggs [1,2].
Triploidy on fish is characterized by changing the
set normal diploid number (2n) of chromosomes
in the state of triploidy (3n). Triploidy can be
achieved by inducing thermal shock, by pressure
shock, different pH, different concentrations of
chemicals, etc. [3‐9].
Polyploid cells have in addition one or more sets
of chromosomes; therefore will have a larger
nucleus. The ratio between the cytoplasm and the
nucleus is constant, so that if the volume of the
nucleus increases the cytoplasm volume will
increase to, and the cell will be larger.
Theoretically, triploid cells will have higher
dimensions than diploid cells. Increased cell size

* Corresponding author: Bencsik Ioan,
0040256277102, [email protected]
113
Bencsik I., et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
Triploid fish (3n) are sterile, but diploid fish (2n)
are not. Triploidisation is carried out in the early
stages after fertilization by thermal shock, osmotic
hyper pressure, electric shock, preventing removal
of the second polar world.
The smear is correctly stained if the final color is
pink or red-violet, if the color is blue or violet the
smear was left for a too long period in contact
with the dye. Microscopic examination of stained
blood smears was made using immersion
objective.
2. Materials and methods
2.2. Triploidy induction
Rainbow trout reproduction cycle starts when the
water temperature reaches 8-10°C; those are the
natural conditions which are necessary to induce
this process and our experiments were performed
under these conditions. Eggs were fertilized by
using the wet method. Collected eggs from several
females were placed in a vessel and then the
semen (for eggs that are collected from one female
is necessary the semen to be collected from 2
males). Afterwards, the eggs and the semen were
mixed in the vessel.
After homogenization, the vessel was placed in
water for temperature equilibration, water that was
used for semen activation. After 5 minutes from
sperm activation and eggs fecundation, thermal
shock was induced to retain the second polar body
inside the fertilized egg and for triploidy
generation. Thermal shock was realized in 28°C
water. To induce the thermal shock we have used
a thermostatically controlled water bath from
stainless steel. Thermal shock was applied for 510 minutes. After that the eggs were counted and
incubated. We have obtained and counted
approximately 70.000 eggs after the thermal
treatment. Eggs from the control group after
fecundation were put directly to incubation step.
During the incubation period dead eggs, white
colored, have been removed on a daily basis and
counted through all 38 days incubation period.
During the whole period of incubation until
hatching 340°C were summed. For statistical
interpretation of the obtained data and graphs
preparation a special program was used.
2.1 Blood smear preparation technique
Blood smear is prepared following the next steps:
blood sampling, spread the blood drop on the
slide, fixation and staining.
Blood sampling:- blood is collected by cardiac
puncture, first drop is removed, the second drop is
collected on clean slide. Afterwards the blood
drop is spread by using another slide (spreader).
The spreader is placed at a 45° angle and backing
into the drop of blood. The spreader catches the
drop and it spreads by capillary action along its
edge, then by a quick and smooth motion blood is
stretched from one to the other end of the slide.
First fixation (physical fixation) was done
immediately by shaking the slide in the air to
avoid the deformation of the erythrocytes.
Staining was done using the panoptical
Pappenhein method. In this method MayGrünwald and concentrated Giemsa (4-10%)
solutions were used.
Staining was done by performing the following
steps:
Slide with the blood smear was placed on
a support in a Petri dishfrotiul, and May-Grünwald
solution was poured; smear was left for 2-5
minutes in contact with May-Grünwald solution
(chemical fixation)
To remove May-Grünwald the same
number of bidistiled water were pored and left on
for 1-3 minute.
The dye was removed without washing
the slide and then the smear was covered with
Giemsa solution. Blood smear was left in contact
with Giemsa solution for 20-40 minutes. During
this step was strengthening the staining of
acidophilic, neutrophile and basophile leukocyte
components colored with May-Grünwald solution
and the azurofile granulations in lymphocytes and
monocytes.
In the end the Giemsa solution was
removed and the smear was washed with water
and then placed on a support and left there to be
dryed on the air.
3. Results and discussion
To establish the triplody induction rate in rainbow
trout embryos we started from the idea that
thermal shock applied after 5-10 minutes after
fertilization will block the elimination of the
second polar body (2n + n = 3n). From the
experimental group formed from triploid trout we
have chosen randomly 30 specimens that were
114
Bencsik I., et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
slaughtered. After slaughtering the blood was
collected and the smears prepared.
Erythrocytes were examined on blood smears, on
minimum 10 visual fields. To determine the
exact dimensions for erythrocytes we measured
both, cell area and cell perimeter. Results from
these measurements done on triploid trout are
presented in Table 1. From those data we observe
that the erythrocytes area has an average of 42.44
μm ² with values ranging between 32.7 to 52.8
μm². Erythrocytes perimeter average is 25.40 μm,
with minimum and maximum variation between
21.9 – 29.63 μm.
Figure 1. Eritrocytes from triploid trout
Figure 2. Eritrocytes from diploid trout
Ref. no.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
Table 1. Area and perimeter measurements results of erythrocytes from triploid rainbow trout
Number of
Area
Standard
Perimeter
Standard
measurements
(µm²)
deviation(s)
(µm)
deviation (s)
10
41.5
7.6
24.8
1,7
10
40.5
8.5
24.4
2
10
44.3
6.5
25.9
1.9
10
45.1
8.7
26.6
1.9
10
39.4
5.9
24.5
1.7
10
55.5
9.68
28.3
1.9
10
47.4
12.5
27.4
3.2
10
42.8
10.8
25.5
2.6
10
51.5
11.33
28.3
2.0
10
40.6
6.7
26
2.8
10
44.2
12.5
25.4
4.1
10
33.5
5.9
21.8
2.3
10
46.66
10.12
27.3
1.9
10
36.6
8.1
24.2
2.4
10
47.3
5.8
27
2.0
10
52.8
8.9
28
2.2
10
42.44
9.43
25
1.5
10
49.7
8
25.8
1.9
10
37.6
8.3
24.5
2.4
10
35.22
8.01
23.1
1.9
10
37.7
8
23.7
2.5
10
53.55
9.08
27.2
2.2
10
38.6
7.8
24.5
2.7
10
38.5
10.2
23.8
3.1
10
51.9
9.3
28
2.3
10
58.2
5.7
29.6
1.4
10
39.4
6
25.1
2.2
10
32.7
6.6
21.9
2.1
10
38.5
8.6
23.7
2.0
Average
42.44
8.43
25.40
2.23
115
Bencsik I., et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
Table 1 data were statistically reworked with a
special PC program and the results are presented
in Table 2.
Table 2. The statistical interpretation on area and perimeter on tetraploid rainbow trout erythrocytes
Ref. No.
Specification
x¯ ±sx
Standard
Variance
Coefficient of
deviation
(s²)
variation
(s)
(C.V.)
1
Aria
42.44±1.26
6.8
46.27
16.02
2
Perimeter
25.40±0.36
1.9
3.82
7.4
the coefficient of variation has a value of 7.4
which represents a small variation of this
character.
In case of control group the measurements for area
and perimeter are of erythrocytes from diploid
rainbow trout are presented in Table 3. We can
observe that the average of the area measurements
is 24.10µm² and the perimeter 19.30µm.
From the Table 2 data can be observed that the
area has an average and error of the mean value of
42.44 ± 1.26 μm ², standard deviation has a value
of 6.8, and coefficient of variation is 16.02; which
means that our measurements have medium
variability. In case of erythrocytes perimeter,
media and medium error of the mean was 25.40 ±
0.36 μm, standard deviation had a value of 1.9 and
Table 3. Results of area and perimeter of erythrocytes from diploid rainbow trout.
Ref. no.
Number of
measurements
Aria
(µm²)
Standard
deviation(s)
Perimeter
(µm)
Standard deviation
(s)
1
2
3
4
5
6
7
10
10
10
10
10
10
10
16.7
20.9
29.4
26.3
24.1
30.7
17.5
24.10
2.2
6.9
10.7
8.5
10.1
2.3
5.9
6.65
15.6
17.2
21.4
20.1
19.3
21.8
16.1
19.30
1.1
1.1
1.7
1.2
0.6
0.7
1.1
1.01
Average
Tabel 4. The statistical interpretation on area and perimeter on diploid rainbow trout erythrocytes
Ref. No.
Specification
x¯ ±sx
Standard
Variance
Coefficient of
deviation
variation
(s)
(s²)
(C.V.)
1
Aria
24.10±2.09
5.53
30.61
22.94
2
Perimeter
19.30±0.94
2.50
6.29
12.95
observe that the difference between area
measurements in erythrocytes from triploid trout
and the erythrocytes from diploid trout is
18.34µm²; which shows that erythrocytes from
triploid individuals are having a higher area by
1.76 than diploid individuals.
The difference observed on erythrocytes perimeter
from triploid and diploid individuals is 6.1μm,
which shows that erythrocytes from triploid trout
are having a higher perimeter by 1.31 than diploid
individuals.
From the Table 4 data can be observed that the
area has an average and error of the mean value of
24.10 ± 2.09 μm ², standard deviation has a value
of 5.53 and coefficient of variation is 22.94, which
means that our measurements have high
variability. In case of erythrocytes perimeter,
media and medium error of the mean was 19.30 ±
0.94µm, standard deviation had a value of 2.5 and
the coefficient of variation has a value of 12.95
which represents a medium variation of this
character. From the above presented data we can
116
Bencsik I., et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
5. Dunham, R.A., Aquaculture and fisheries
biotehnologie, Genetic approaches, CABI Publishing,
Cambridge, USA, 2004.
6. Haffray, P. et al., Gonad development, growth,
survival and quality traits in triploids of the protandrous
hermaphrodite gilthead seabream, Sparus aurata (L.).
Aquaculture, 2005, 247: 107-117.
7. Kalbassi, M., Johari, S.A., A study on the production
possibility of all-female triploid rainbow trout
(Oncorhznchus mikiss), J.Sc.& Technol.Agric.
&Natur.Resour., 2008, 12, 44.
8. Kapuscinski A., R., Miller L., M., Genetic guidelines
for fisheries management, University of Minesota,
2007, p.66-68.
9. Quillet, E., Foisil, L., Chevassus, B., Chourrout, D.,
Liu, F.G., Liu, F.,G., Production of all-triploid and allfemale brown trout for aquaculture, Aquat.living
Resour., 1991, 4, 27-32.
10. Benfey, T.J., Sutterlin, A.M. and Thompson, R.J.,
Use of erythrocyte measurements to identify triploid
salmonids, Canadian Journal of Fisheries and Aquatic
Sciences, 1984, 41, 980–984
11. Garcia-Abiado, M.A.R., Dabrowski, K.,
Christensen, J.E., Czesny, S. and Bajer, P., Use of
erythrocyte measurements to identify triploid saugeyes.
North American Journal of Aquaculture, 1999, 61,
319–325
12. Colihueque, N., Iturra, P., Diaz, N., Veloso, A.,
Estay, F., Karyological analysis and identification of
heterochromosomes in experimental gynogenetic
offspring of rainbow trout (Oncorhynchus mykiss,
Walbaum) Rev.Brasil.Genet, 1992, 15, 3, 535-546.
13. Quillet, E., Aubard, G., Queau, I., Mutattion in a
sex-determining gene in rainbow trout Detection and
genetic analysis, The American Genetic Association,
2002, 93:91–99
14. Phillips, R.B., Zajicek, K.D., Ihssen, P.E. and
Johnson, O., Application of silver staining to the
identification of triploid fish cells., Aquaculture, 1986,
54, 313–319.
15. Strunjak-Petrovic,I., Coz-Rakovac, R., Topic
Popovic, N., Micronucleus occurrence in diploid and
triploid rainbow trout (Oncorhynchus mykiss Walbaun),
Vet. Med. - Czech, 2003, 48, (8), 215-219.
4. Conclusions

Erythrocytes area measured on triploid
and diplod individuals can represent an indicator
in establishing the polyploidy grade.

Erythrocytes perimeter measured on
triploid and diplod individuals can represent an
indicator in establishing the polyploidy grade.

Erythrocytes area at triploid individuals,
measured in µm², is 1.76 bigger than in diploid
trout.

Erythrocytes perimeter at triploid
individuals, measured in µm, is 1.31 bigger than
in diploid trout

Coefficient of variation in area
measurement case is medium; this means that this
measurement method has an acceptable precision
and accuracy to be used in determination of the
poliploidy grade in rainbow trout.

Coefficient of variation in perimeter
measurement case is low; this means that this
measurement method has an acceptable precision
and accuracy to be used in determination of the
poliploidy grade in rainbow trout.
References
1. Peruzzi, S., Chatain, B. Pressure and cold shock
induction of meiotic gynogenesis and triploidy.
Aquaculture, 2000, 189: 23-37.
2. Sellars M.J., Coman F.E., Degnan B.M., Preston
N.P., The effectiveness of heat, cold and 6dimetilaminopurine shoks for inducing tetraploidy in
the kuruma shrimp, Marsupanaeus Japonicus (Bate),
Journal of Shellfish Research 2006, 25 (2),631-637.
3. Beaumont, A., Hoare, K., Biotehnology and genetics
in fisheries and aquaculture, Blakwell Publishing, 2003
4. Bohl, M., Zucht und Production von
Suβwasserfischen, Verglas Union Agrar, 1999, p. 555558.
117