AND Pangasius nasutus - JSSM - Universiti Malaysia Terengganu

Journal of Sustainability Science and Management
Volume 6 Number 1, June 2011: 28-35
Introduction
Catfishes of the family Pangasiidae, such as
Pangasianodon hypophthalmus (Sauvage, 1878)
(Tarnchalanukit, 1985), are of great economic
importance in Southeast Asia region, including
Vietnam (Hung et al., 1999). Thus, many
attempts in breeding, including crossbreeding,
have been done to increase their production
in aquaculture practices. Crossbreeding is the
mating of individuals of different species,
varieties or lines. This process combines the
best of each individual breed and its genes into a
more heterozygous animal (Burnside, 2004) and
the offspring is known as a hybrid. Hybrids show
characteristics of both parents and when a hybrid
ISSN: 1823-8556
© Universiti Malaysia Terengganu Publisher
CROSSBREEDING OF Pangasianodon hypophthalmus (SAUVAGE, 1878)
AND Pangasius nasutus (BLEEKER, 1863) AND THEIR LARVAL
DEVELOPMENT
ANUAR HASSAN * , MOHD. AZMI AMBAK AND AGUS PUTRA A. SAMAD
* Institute of Tropical Aquaculture (AKUATROP), Universiti Malaysia Terengganu (UMT), Mengabang Telipot, 21030 Kuala
Terengganu, Terengganu, Malaysia.
*Corresponding author: anuar@umt.edu.my
Abstract: This study was conducted to determine the feasibility of producing hybrid catfish through
reciprocal crossbreeding between Pangasianodon hypophthalmus (Sauvage) female and Pangasius
nasutus (Bleeker) male (HN) and between P. hypophythalmus male and P. nasutus female (NH).
Two control groups with homogenous breeding of P. hypophthalmus (HH) and P. nasutus (NN)
were also conducted. The results of HN showed better breeding performance with an average value
of fertilisation (81.17%), hatching (83.06%) and deformation rate (8.51%) compared to NH with
60.96, 54.61 and 14.68%, respectively, for the same parameters. For the control HH, fertilisation,
hatching and deformation rate were 93.18, 89.73 and 4.14%, respectively, while, for NN, 76.83,
68.26 and 7.16%, respectively. Fertilised eggs of HN (1.08-1.10 mm) were smaller than NH (1.80-
1.90 mm), with no significant difference with their parental fishes (HH and NN). The incubation time
of HN (20-26 hours) was shorter than that of NH (30-36 hours) after fertilisation. The incubation
times of HH and NN were: 18-24 and 36-40 hours, respectively. Newly-hatched larvae of HN
were: 3.92±0.04 mm in total length and 0.22±0.02 mg in weight, while, NH were 5.69±0.08 mm
and 1.76±0.07 mg, respectively. Newly-hatched larvae for control (HH) was 3.47±0.12 mm long
(TL) and 0.22±0.03 mg weight, while for NN was 5.79±0.26 mm and 2.25±0.59 mg, respectively.
Thus, it could be concluded that cross-bred HN showed relatively better performance in the crossbreeding
programme.
KEYWORDS: crossbreeding, Pangasianodon hypophthalmus, Pangasius nasutus, embryonic and
larval development
Received: 03 February 2010 / Accepted: 27 February 2011
shows characteristics superior to both parents,
it is said to have hybrid vigour or positive
heterosis, which, of course, is the ultimate
goal of breeding (Dunham and Masser, 1998).
Moreover, crossbreeding also has been used
to produce hybrids that have improved growth
rates, survival rates, lower food conversion rates,
improved disease resistance, etc. (Tave, 2003).
Although there are many literatures available on
the reproductive behaviour of different catfish,
spawning strategies and breeding techniques, the
literature on the early developmental stages of
hybrid catfish is still limited. There have been
similar studies on the crossbreeding technique
of Clarias sp. (Na-Nakorn et al., 1993; Morni,
2003), morphometric and biometric analysis of

Anuar Hassan et al 29
Pangasius sp. (Gustiano, 2004) and rearing trial
of Pangasius hybrid (Adi, 2005).
Pangasius nasutus (Bleeker, 1863) is an
excellent food fish with very white fine-grained
and sweet flesh, but it is still quite difficult to
find in the market because the availability is still
dependent on catches from the wild, which has
led to a reduction in the natural resources. Beside
that, this fish is a slow-growing fish and the price
of this fish is very high, almost three times that
of P. hypophthalmus. P. hypophthalmus is a fastgrowing
omnivorous fish and hence a potential
candidate for culture in ponds and cages. It
accepts a wide range of artificial feeds or diets,
and is also quite hardy and resistant to diseases.
The major constraint of P. hypophthalmus is
related to its marketing part, whereas P. nasutus
with its whitish flesh is preferred over the yellow
flesh of P. hypophthalmus, not only in Malaysia
but also in other markets such as Asia, Europe
and North America which constitute potential
export destinations.
The present study is aimed to determine
the feasibility of producing hybrids through
reciprocal crossbreeding of P. hypophthalmus
female and P. nasutus male (HN) and P.
hypophythalmus male and P. nasutus female
(NH). A determination of the embryonic and
larval development of their hybrid was also
conducted.
Materials and Methods
Breeding Programme
Matured broodstocks of both species were not
fed 24 hours prior to capturing and handling.
Fully-matured broodstocks were induced using
a combination of hormones with HCG (human
chorionic gonadotropin) at a dose of 500 IU/kg
female body weight and ovaprim (commercial
mix of GnRh and Domperidone) given 24 hours
after the HCG injection at a dose of 0.6 ml/kg
female body weight. In order to increase the
quantity of collected semen and reduce viscosity,
males received a single ovaprim injection of 0.4
ml/kg given at the same time as the ovaprim
injection of females was given. Intramuscular
J. Sustain. Sci. Manage. Volume 6 (1) 2011: 28-35
injection was done below the dorsal fin. After
hormone injection, broodstocks were placed in
separate tanks to complete the spawning process.
Four breeding programmes were conducted
in this study, which were marked as HN for
mating of P. hypopthalmus female X P. nasutus
male, and NH for mating of P. nasutus female
X P. hypopthalmus male. HH for mating of
P. hypopthalmus X P. hypopthalmus; NN for
mating of P. nasutus X P. nasutus. Based on
these breeding programmes, NH and HN were
the reciprocal crosses, whereas NN and HH were
identified as control. Three replicates were used
for each of the crossbreeding programme. The
artificial fertilisation technique used in this study
was the dry method. The sperm was spread over
and mixed manually with collected eggs. Both
eggs and sperm were mixed gently with a feather
until the sperm was homogenously spread in the
eggs mass. Fresh water was added quickly in
order to activate all sperm at the same time. Eggs
and sperm were mixed again for one minute to
ensure the maximum fertilisation. Then, eggs
were rinsed with clean fresh water to remove
excess of sperm before transferring them into
incubators for incubation. After fertilisation
and before stocking them into incubators, eggs
underwent a treatment with clay to reduce their
stickiness. It was done by pouring the clay
suspension to the collected fertilised eggs and
delicately mixing the eggs and the clay using
a feather for about 5 minutes until they do not
adhere to each other. Then, the eggs were rinsed
to remove the excess clay and ready to be placed
in incubators. In this study, eggs were incubated
in running water funnel (MacDonald jar).
Before stocking eggs into their incubator, water
flow was stopped for a while in order to avoid
loss of eggs through the water outlet. During
the incubation period, water qualities were
monitored, including constant water flow and
temperature. On the day of hatching, larvae were
counted and transferred into the experimental
fibreglass tanks.
Parameters such as fertilisation, hatching
and deformation rate were calculated following
the formula by Gunasekara et al. (1996):

CROSSBREEDING OF Pangasianodon hypophthalmus (SAUVAGE, 1878) 30
No. of eggs fertilised
Fertilisation Rate = X 100
No. of total eggs
No. of eggs hatched
Harching Rate = X 100
No. of fertilised eggs
No. of deformed larvae
Deformation Rate = X 100
No. of larvae hatched
Embryonic and larval development
The development of the embryo was checked
soon after fertilisation. To check the embryonic
development, around 100-300 eggs were
transferred to transparent buckets. The water in the
buckets was changed regularly to reduce fungal
development and separate the eggs shells (from
dead eggs) from the fertilised eggs. Furthermore,
the eggs were monitored regularly according
to the time of embryonic development using a
microscope at magnification of 25 X and the
eggs were measured using a micrometer. Larval
development studies covered the morphological
characteristics of the larvae until juvenile stages.
500-litre fibreglass rearing tanks were used
for the experiment. Daily observations during
the first week after hatching (wAH) followed
by once a week interval until six weeks were
conducted. Without any fixative solution, soon
after sampling, the larvae or juveniles were
directly observed under microscope after being
anaesthetised by MS222 (tricaine methane
sulfonate) at a concentration of 5-10 ppm. During
the larval stage, development of the new hybrids
was observed under a microscope. Total length
was measured using a micrometer, where, during
juvenile stage, the total length was measured
using a digital caliper (Tresna, USA) and the body
weight by using an analytical balance.
J. Sustain. Sci. Manage. Volume 6 (1) 2011: 28-35
Results
The breeding performance of reciprocal
crossbreeding between P. hypophthalmus and
P. nasutus in three trials are shown in Table
1. Results of fertilisation rate of HH showed
the best result (93.18±2.12%) followed by
HN (81.17±6.37%) and NN (76.83±1.30%).
Statistical analysis showed that fertilisation rate
of (HN) crossbreeding was not significantly
different (p>0.05) with NN mating, although
significantly lower compared with (HH)
mating. However, NH showed the poorest result
(60.96±1.72%) and was significantly different
with all other groups.
Similar to the results of fertilisation rate,
hatching rate of HH mating also showed the
best result (89.73±1.59%) followed by HN
(83.06±2.74%) and NN (68.26±1.43%) mating.
Statistical analysis showed that average hatching
rate from each mating was significantly different.
Meanwhile, NH mating showed the poorest
result with only 54.61±3.11% of fertilised eggs
being hatched.
In sequence of the poorest result of
fertilisation and hatching rates, NH showed
the highest (14.68±2.66%) deformation rate
compared with other matings. The averages
of deformation rate from other matings were
consistently low and not significantly different
from each other. The deformation rate of (HH),
NN and HN were 4.14±1.05, 7.16±3.88 and
8.51±1.63%, respectively.
Incubation period
Observation of 50 oocytes from selected
broodstocks, showed that P. hypophthalmus became
sexually matured when oocytes diameter reached
1.00 mm. whereas for P. nasutus it was 1.60 mm.
Table 1. Mean (±sd) fertilisation, hatching and deformation rate of P. hypophthalmus (HH),
P. nasutus (NN) and their reciprocal hybrid (HN and NH).

Anuar Hassan et al 31
The incubation time of HN (20-26 hours)
was shorter than that of NH (30-36 hours) after
fertilisation. The incubation time of HH and NN
were 18-24 hours and 36-40 hours, respectively.
Larvae were not hatched in synchronisation but
spread over a period of several hours.
Embryonic development
After water contact, eggs were subjected to
a rapid hydration leading to the formation
of the perivitelline space (Fig. 1A). The egg
mucous coats also swelled in contact with water
and became adhesive. At this stage, whether
fertilised or not, the swollen eggs had a yellowish
colour. However, fertilised eggs soon started to
develop and the first cleavage (stage two-cells)
became clearly visible after 30 to 35 minutes
of fertilisation followed by 4 cells (Fig. 1B), 8
cells and 16 cells stages at 40 to 90 minutes after
fertilisation. The eggs were in the morula stage at
about 2 hours after fertilisation (Fig. 1C) and then
cells were in the blastula stage at 3 to 6 hours after
J. Sustain. Sci. Manage. Volume 6 (1) 2011: 28-35
fertilisation. Shortly thereafter, the gastrula stage
started and the cell division progressed to cover
the yolk mass. The last step of the gastrula stage
occurred about 9 to 9.5 hours after fertilisation
(Fig. 1D) and it was characterised by the closing
of the blastopore. Further, 10 to 10.5 hours after
fertilisation, the embryo appeared in the form of
a half ring with the head and the tail buds present
at the two ends. Afterwards, the first segment of
the body became rapidly visible while the tail bud
started to grow longitudinally and the embryo
starts to twist its tail occasionally. Compared to
HN (Fig. 1E) embryo, NH showed developments
of an earlier dark pigmentation which even
appeared before hatching (Fig. 1F). Movements
of the embryo became more and more active until
hatching occurred (Fig. 1G and Fig. 1H).
Morphological development and behaviour
changes
Newly-hatched larvae of HN larvae had an
average total length of 3.29±0.17 mm and body
Figure 1. Embryonic development; A. Fertilised egg; B. 4-cell stage; C. Morulla stage; D. Closing
of blastopore stage; E. HN active-moving embryo; F. NH active-moving embryo; G. Newly-hatched
HN hybrid larvae; H. Newly-hatched NH hybrid larvae.

CROSSBREEDING OF Pangasianodon hypophthalmus (SAUVAGE, 1878) 32
weight of 0.22±0.07 mg. Whereas, (NH) larvae
an average total length of 5.69±0.17 mm and
body weight of 1.76±0.25 mg. Dark pigmentation
spots appeared at the base of the yolk sac at 6
hours after hatching (hAH) with NH. Together
with this stage, the fins formation started. At this
stage, their tail was always beating but still no
sign of movement of the body. Their morphology
gradually changed, at 18 hAH with the formation
of mouth together with the formation of pairs of
short and thick barbels, and, at the same time, the
eyes were clearly pigmented (Fig. 2I and Fig. 2J).
Then, two pairs of barbels clearly appeared in
each side of the upper and lower jaws at 24 hours
after hatching. Corresponding with the gradual
decrease of yolk size, the intestinal tracts began to
move during 48 hAH. After that, the digestive tract
gradually developed along with the development
of barbels associated with feeding behaviour. At
54 hAH, the artemia naupli given were found in
the intestinal tract of larvae showing the start of
feeding (Fig. 2K).
J. Sustain. Sci. Manage. Volume 6 (1) 2011: 28-35
For all the stages of larval development,
although the fin rays started to form at 24 hAH
which started at the caudal side, the division of
specific fins were still undifferentiated. The clear
formation of the pectoral fin, paired pelvic fins
and the rudiments of caudal fin newly appeared
at 1 wAH (Fig. 2L and Fig. 2 M). At this stage,
dark pigmentation covered the whole body of
the hybrid larvae. Further observation showed
the body colour changed between 1 wAH and 2
wAH, whereas, the abdomen appeared slightly
whiter than before. Moreover, at 6 wAH (Fig. 2
N and Fig. 2O), all parts of the body were fully
formed and hybrid larvae were morphologically
like adult fish. At this stage, (HN) larvae had an
average total length of 59.60±1.96 and NH had an
average total length of 56.29±1.25.
Discussion
Fertilisation and hatching rates of the fish of the
same parents (control) were compared with the
previous results. As a comparison, fertilisation
Figure 2. I. HN larvae; J. NH larvae; K. HN larvae ingested artemia nauplii; L. HN larvae (1 wAH);
M. NH larvae (1 wAH); N. HN juvenile (6 wAH); O. NH juvenile (6 wAH).

Anuar Hassan et al 33
rate of HH obtained in this study (93.18±2.12%)
was not different from the reports by Sularto
(2005) which reported 95.09±2.68%, but the
hatching rate for the current study (89.73±1.59%)
was higher (75.16±2.86%). It showed that
the temperature (29-30 o C) affected a good
performance of the hatching rate. According to
Saidin and Othman (1986), the incubation period
for 24-26 hours at a water temperature of 28-
32 o C; fertilisation occurred between 70-80% and
with 30-45% hatching rate.
In this study, crossbreeding between P.
hypophthalmus and P. nasutus produced the
hybrid. The fertilisation and hatching rate of
P. hypophthalmus hybrid larvae (HN) were;
81.17±6.37% and 83.06±2.74%, whereas P.
nasutus hybrid larvae (NH) were 60.96±1.72%
and 54.61±3.11%, respectively. There are several
other reports of the successful crossbreeding of
Pangasiid, for example, Adi (2005) showed that, the
crossbreeding between female of P. hypopthalmus
and male of P. djambal was successful with a lower
hatching rate (29.82±15.02%). In crossbreeding
between Clarias and Pangasius, fertilisation was
also very high (68-97%), but hatching rate was
reduced between 11 and 23% (Tarnchalanukit,
1985). In crossbreeding among clariid, (C.
macrocephalus X C. gariepinus) the fertilisation
rate reached 48-83% with a varying hatching rate
of 40-86% (Morni, 2003).
On the other hand, crosses between P.
hypophthalmus female X P. nasutus male (HN)
showed a high performance, compared with P.
nasutus female X P. hypophthalmus male (NH),
Beside the lowest fertilisation and hatching rate, it
also had a higher deformation rate (14.68±2.66%)
than any another group. In order to classify the
success level of any crossbreeding programme,
breeding performance such as: fertilization rate,
hatching rate, deformation rate and the survival
rate of hybrid progeny, comparison with their
parental species (control) were commonly used
(Chevassus, 1983).
From the current study between the
reciprocal crossing, crossbreeding between
P. hypophthalmus female X P. nasutus male
(HN) was feasible in crossbreeding programme
J. Sustain. Sci. Manage. Volume 6 (1) 2011: 28-35
between these pangasiid. Similar results
were found in crossbreeding of other fishes.
For instance, Morni (2003) reported that
crossbreeding between Asian and African catfish,
only crossbreeding between C. macrocephalus
female X C. gariepinus male was feasible;
furthermore, only crossbreeding between C.
gariepinus X H. longifilis (Legendre et al., 1992)
and crossbreeding between Catla catla X Labeo
fimbriatus (Basavaraju et al., 1995) was feasible.
However, for several crossbreeding
programmes such as crossbreeding between
Oncorhynchus. ksutch, Salmo gairdneri and
S. trutta studied by Chevassus (1983) it was
found that, crossbreeding was only achieved in
the single-way crossing between S. gairdneri
female X O. ksutch male, S. gairdneri female
X S. trutta male and S. trutta female X O.
ksutch male. In case of grouper, Tseng and Poon
(1983) reported that crossbreeding between
white-spotted green grouper Epinephelus
amblycephalus female X red grouper E. akaara
male were the only possible cross. In the current
study, crossbreeding between both species was
a success. After mixing sperm and egg of both
species, fertilised eggs soon started developing
and the first cleavage (stage two-cells) became
clearly visible at 30 to 35 minutes. This
successful fertilisation was similar to crossbreeds
of Mytilus edulis and M. trossulus as reported by
Toro et al. (2006). Mature fertilised eggs after
cross mating of parental fish were circular and
adhesive in nature, whereas mature unfertilised
eggs were elastic and spherical in shape. The egg
capsule and yolk sphere were yellowish-brown
in colour. Fish from the same catfish family
(Clarias batrachus) had a greenish egg capsule
(Mookerjee and Mazumder, 1950). Fertilised
eggs of P. hypophthalmus female and milt from
P. nasutus male (HN) were between 1.08 and
1.10 mm and fertilised eggs of P. nasutus female
and milt from P. hypophthalmus male (NH)
were between 1.80 and 1.90 mm in diameter. In
comparison, diameter of fertilised eggs of both
these matings were bigger than other native
catfishes, whereas, the diameter of fertilised
eggs of Clarias gariepinus are 0.6-0.8 mm (Zaki
and Abdulla, 1984) and diameter of fertilised

CROSSBREEDING OF Pangasianodon hypophthalmus (SAUVAGE, 1878) 34
eggs of Heteropneustes fossilis were 1.0-1.2 mm
(Bhargava, 1970). However, they were smaller
than eggs diameter of Channel catfish (Ictalurus
punctatus), whereas, the egg diameter was 3.5-4
mm (Scott and Crossman, 1998).
Embryonic development stages of mating
between P. hypophthalmus female X P. nasutus
(HN) required 20-26 hours, while, P. nasutus
female X P. hypophthalmus male (NH) were
30-36 hours until hatching. It showed that the
gametes (milt) play a big role in the duration
of embryonic development. Early embryonic
development of C. gariepinus requires 21-26
hours (Bruton, 1979). The incubation period of
C. batrachus needs to be 17-22 hours (Thakur
et al., 1974). The (HN) embryo showed twisting
movements inside the egg shell 1-2 hours before
hatching, while (NH) embryo was 6-7 hours
before hatching. This magnificent twisting
movement has also been reported in other
catfishes like, H. fossilis (Thakur et al., 1974),
C. gariepinus (Zaki and Abdulla, 1984) and C.
batrachus (Thakur, 1980). Newly-hatched HN
and NH larvae ranged from 3.20-3.50 mm and
5.50-5.70 mm, total length, respectively. Newlyhatched
C. gariepinus measured 3.4-4.0 mm
(Peteri and Nandi, 1992) and C. batrachus were
5.8 mm in length (Mookerjee and Mazumder,
1950).
Hybrid larvae (NH) hatched with dark
pigmentation since early hatching. They were
different from hybrid of C. macrocephalus X
C. gariepinus, where, the hybrid hatched as
transparent without any pigment (Morni, 2003).
From the observation, development of
hybrid larvae was relatively fast compared to
other studied species. The jaw began to move at
18 hours after hatching and, at the same time,
the eyes were deeply pigmented, and the barbels
started to develop. As a comparison, the eyes of
marble goby would only be pigmented 4 days
after hatching and their jaw stated to move at
2-3 days after hatching (Senoo et al., 1994).
At 1 wAH an average total length of 7.34±0.39
mm and 13.15±0.50 mm, were reported with
HN and NH larvae, respectively. Morni (2003)
reported that the larval period of hybrid catfish
J. Sustain. Sci. Manage. Volume 6 (1) 2011: 28-35
(C. macrocephalus X C. gariepinus) was 21
days after hatching. Further, observations found
that morphological performance of the hybrids
were like the adult 6 wAH, with an average total
length of 59.60±1.96 mm and 56.29±1.25 mm,
for HN and NH larvae.
Acknowledgement
The authors are grateful for technical contribution
of staff members of LRPTBPAT, Sukamandi.
Special thanks due to Mr. O. Charman, Mr.
Sularto, R. Utami and Mr. Pamungkas, W. for
their help in various study aspects.
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