Triploid was induced in African Catfish (Heterobranchus longifilis) by cold shocking activated eggs at 5 degree C for forty minutes starting 3-4 minutes after fertilization. Triploidy was confirmed from mitotic chromosomes prepared from embryo which showed 100% triploidy in the cold shocking treatment and 100% diploidy in the control treatmen

Aluko, P.O.

Olufeagba, S.O.

Omotosho, J.S.

Oyewole, S.O.

Raji, A.

English

Aquatic Commons

INTRODIJCTION
One of the current ntethods of improving growth
performance of aquacultural species is through
biottelmology approach. This could be through
hybridization. chromosome manipulation, gene
manipulation etc. Chromosome manipulation may
be in .form of Triploidy induction. This method
involved the inhibition of extrusion of the second
polar body during the second meiotic division
(Thorgard. 1983).
Many authors have published instances of
successful triploidy induction by cold shock in
different species in both freshwater and marine
species (Chao et al., 1986: Tave_ 1994: Manickam,
1990: Olufeagba et al: 1996).
longifilis potentials- as a good aquacultural
spez,-ies has been described (Teugels. et al. 1990).
The karyomorphology is also known. (Olufeagba
press) but a thorough study of the early survival
and growth of triploid compared with the control
has not been reported. Therefore. in this study.
ABSTRACT
Trip toidy was induced o African Catfish (Hetembranchus longrfilis) by coid shocking activated eggs at 5°C for forty
minutes starting 3 - 4 minutes after fertilization.
Triploidy was con:finned from mitotic chromosomes prepared from embryo which showed 100% triploidy in the cold
shocked treatment and 100% diploidy in the control treatment.
247
longifilis triploids was induced by applying cold
shock at 5°C for fort.y minutes in cold chamber on
3-4 minutes old artificially activated eggs.
MATERIALS AND METHOD
Breeders weighing between 250-500 grams were
collected from the wild from Anambra. State of
Nigeria (5° latitude). They were transported to
National Institute for FreshwaterFisheries Research
(NIFFR)New Bussa 10m x 10m x 1.5m3 outdoor
concrete tanks for them to acclimatize. They were
fed with 40% crude protein. NIFFR feed. During
breeding, male with protruded reddish genitalia wa s
choosing and a female that released free eggs on
gentle strolling of fingers on the be,lly.
The breeders were injected with oue dose of 0.5 ml
per kilogram of fish. The latency period before
stripping was 15 hours at 26°C (±1). After fifteen
hours, the male was sacrificed and the testes were
removed. The eggs were hand-stripped and were
fertilized with the sperm from the testes.
TRIPLtIIIY I IUCTIO,N IN Heterobranchus longifilis
(FAMILY: CLA IDAL) BY COLD SHOCK
S.O. 0 UFEAGBA1, P.O. ALUK02,I.S. OMOTOSII03,
S.O. OYENVOLE3 and A. RAO
'Federal College of Freshwater Fisheries Technology,
PMB 1500, New Bussa, Niger State
'Arational Institute for .Freshwater Fisheries Research,
PtI,LB 6006, Are)", Bussa, Afiger State.
'Biological Sciences Department,
University of liorin, Kwara State.
TREPLOIDY INDUCTION
Cold shock treatment of the fertilized egg was done
inside a cold chamber maintained at 5°C for 40
minutes. The fertilized eggs were distributed into
four beakers. Kakabans were put in the bealcers for
the eggs to adhere to them. Cold shocking started
4 minutes after fertilization.
One 60cm x 30cm x 30cm3 aquaria served as
control as the eggs were allowed to develop
normall3r under room temperature (25°C ±1).
After 40 minutes of cold treatment, the eggs were
transferred to 60cm x 30cm x 30cm3 aquaria with
aerated water at room temperature.
RES ULTS
Percentagehatchability was calculatedbased on the
hatchlings at twenty four hours. Survival rate was
monitored for fourteen days and analysis of
variance was used to find significance difference at
5% between the control and triploid survival. After
two weeks of monitoring in glass aquaria, the fry
were transferred to net hapa fixed in 4mx4m x 1.2
cri3 earthen pond. Fish were fed once daily with
40% crude protein feed. Fry have access to natural
zooplankton, as the pond were regularly fertilized
with chicken droppings.
ASSESSMENT OF PLOIDY LEVEL
Triploidy induction was confirmed from 12 hours
old fry that was allowed to swim in 0.001%
colchicine and 0.5% cobalt chloride solution for
twelve hours, followed by two hours swimming in
Table 1: Percentage fertilization, hatchability and survival in Heterobranchus longififis.
Treatments Number of Number of Number of Number of
Eggs (±10) Fertilized Eggs Hatchlings a b norma 1
(±10) (± 10) embryo
Triploid 350 320 (91.4)
Control 350 315 (90)
*Percentage hatchability in relation to number of fertilized eggs
** Percentage abnormal embryo
248
distilled water. The -fry were then removed and
preserved in fixative made up of one part glacial
acetic acid and tlu-ee parts analytical ethanol. The
fixed hatchlings were kept in the refrigerator until
use.
Trunk regions of 2-3 hatchlings were minced with
fine tipped forceps for one minute to form cell
suspension in a depression slide. 2-3 drops of the
suspension was then placed on warmed slides and
allowed to dry on slide warmer maintained at 40-
45°C. The slides were stained for 30-40 minutes
with F.L.P. orcein stain (formic acid, lactic acid,
propionic acid). The stain was rinsed off and dried
again. The slides were observed using a Zeiss
binocular microscope equipped with a camera. 50
well spread metaphase were examined in both the
triploid and control cells. The mean number of the
chromosome count was taken as the Chromosome
number.
Well spread metaphase spread were photographed
and the negatives were developed and printed.
Ninety fry each in the control and triploid
treatments were distributed in 3 aquaria (30
fry/aquaria) to monitor daily survival for two
weeks, after which the pooled weight and average
length were determined. Fry were fed with
zooplankton collected from NIFFR natural Fish
food production unit.
The results of the hatchability are shown in table 1.
The cold shock eggs had 48.2% hatchability, while
93.2% hatchability was recorded in the control
exp eriment.
154 (48.2)* 7 (4.5)**
293 (93.2)* 2 (0.6)**
In the control treatment, the mean number of
chromosome was 50 number (Plate 1A) while in
triploids, 75 was the average number of
chromosomes (Plate 1B).
Less than 5% of the triploid, and less than 1% of
the control treatments developed some abnormal
characteristics. These include short or curved tail,
distorted. Yolk sack and microcephalic. These
embryo died within three days of hatching.
249
The daily survival rate is shown in Table 2, and
the mean length (cm) and mean weight (grams) are
shown in Figure 2. After 2 weeks, the meani,veight
for the triploids was 0.043gm while for control it
was 0,039 gms. The mean length for Triploids was
1.7cm, while for control, it was 1.5cm.
The number of suwival in Triploid was 74 while,
in the control, 80 survival was recorded after 2
weeks. There was no significant different between
the survival in control and Triploid experiment.
(0.05% cal. F value = 3.416).
Table 2: Survival, Mean length, and Weight of diploid and triploid Heterobranchus longifilis
reared in aquaria tanks.
Treatments Weight (gin) Length (cm) Percentage Survival
Triploid 0.061 1.8 66.1
Diploid 0.041 1.5 91.6
APlate 1: (a) Diploid metaphase chromosome in fieterobranchus longifilis 2n = 50
(I)) Triploid metaphase Chromosome in H. longifilis 3n = 75 (map, x 3260)
250
DISC USS ION
Triploidy was successfully induced in catfish eggs
exposed to cold shock at 5°C for a duration of 40
minutes as shown in plate lb. This experiment still
continue to increase triploid yield from cold
shocking of eggs.
The triploid fish were as viable as the diploid after
hatching. This was clearly shown in the Fishers
(1948) analysis of variance test -which confirmed
no significant difference at both 5% and 10%
signifi.cant levels in the survival of diploid and
triploid after hatching. The low percentage
hatchability is in agreement with previous authors
(Hervai et al 1980; Manickarn, 1991; Linhart et al;
1991; Olufeagba et al 1996).
The goal of triploid induction is to increase the
growth rate above the diploid control (normal).
This has been demonstrated due to lack of sexual
maturation in sterile triploid (Valenti, 1975;
Purdom, 1976). This is expected to be more
pronounced after sexual maturity, however, this has
been shown in this experiment where the mean
weight and mean length of triploid Were higher
than normal group (Table 2). No work on early
growth of Triploid has been published.
The fry are being raised to maturity to record data
on growth, feed efficiency and gonad° somatic
index under earthen condition. Any desirable
feature gained from triploid if. long/fills will go a
long way to increase catfish production.
REFERENCES
Chao, N.II, S.J. Chen; Lino LC, (1986): Triploidy induced by
colcl shock in cyprinid loads;34i.sgor-nu.s. anguilli, audatus:
In the first A.sian Fisheries Forum, J.L. Ivlacteam,
Dizon, and L.V. Hosillos (Editors). Asian Fisheries
Society, manila, Philippines.
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Fisher, R.A. (1948): Statistical methods for research workers.
New York, Oliver and Boyd Inc,
Gervai J.S; A. Nagy; L. Borvath, V. Cysanyl C (1980):
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Unhurt 0; S. '<lido; R. Billard; V.Slechtn; E.V. IVIikodbia
(1995): Morphology, cotnposition and fertilization of
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A/Initial-tin, P. (1991): Triploidy induced by cold shock in the
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Raji (1996): Preliminary report on the growth
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'rave Douglas (1992): Genetics for fish hatchery managers. 2nd
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Tengels, G.G; T. Dennyer and U. Legendre (1990): A
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Thorgard, G.II; (1933): Chromosome set manipulation and sex
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Triploidy induction in Heterobranchus longifilis (Family: Clariidae) by cold shock

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Triploidy induction in Heterobranchus longifilis (Family: Clariidae) by cold shock

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