Original Contributions

Growth Performance and Gonadal Development ofGrowth Enhanced Transgenic Tilapia Oreochromisniloticus (L.) Following Heat-Shock-Induced Triploidy

Shaharudin Abdul Razak, Gyu-Lin Hwang, M. Azizur Rahman, and Norman Maclean*

Division of Cell Sciences, School of Biological Sciences, University of Southampton, Bassett Crescent East,

Southampton SO16 7PX, United Kingdom

Abstract: Triploid induction offers a way of considerably reducing fertility in fish, and could therefore be

employed to help ensure that any adverse environmental impact of transgenic fish was markedly less. In order

to produce sterile growth-enhanced transgenic fish, we have induced triploidy in two lines of transgenic tilapia.

Growth performance and gonadal development were analyzed following triploidization by heat shock. Ploidy

status was confirmed by nuclear size measurement of erythrocytes. Erythrocytes of triploids were found to be

1.5 times larger than diploids. Observations of growth enhancement and gonadal development were made on

diploids and triploids from both transgenic and nontransgenic full sibling batches. In both lines, transgenic

diploids were superior in growth performance, followed by transgenic triploids, nontransgenic diploids, and

nontransgenic triploids. Although the testes of transgenic triploids were significantly smaller than those of

nontransgenic triploids and nontransgenic diploids, histologically they did not show signs of gross deformation.

There were also some spermatozoa present in the testes of some triploids, which could be indicative of

reproductive functionality. However, the ovaries were devoid of oocytes, underdeveloped, and deformed in all

triploids and were completely nonfunctional. Although the best growth performance was shown by the fertile

diploid transgenics, the triploid transgenic females could offer a good option for aquaculture purposes because

they showed superior growth performance over the normal wild-type tilapias with the advantage of sterility to

ensure nonhybridization and noncontamination with the local gene pool. However, careful monitoring of

potential gene flow would be required prior to commercial use.

Key words: Growth enhanced, transgenic fish, tilapia, and triploidy

INTRODUCTION

Transgenic strains of fish offer some clear advantages for

aquaculture (see reviews by Iyengar et al., 1996; Maclean,

1998) but raise problems of environmental impact in the

event of escape or release. One way to reduce or eliminate

this problem is to make such fish sterile through triploid

induction provided that some form of effective contain-

ment was also used. Triploid induction has been used in fish

culture because of its perceived ability both to elevate

growth and, at the same time, induce sterility. By shocking

Received December 1, 1998; accepted May 18, 1999.

*Corresponding author; telephone +44 (0) 1703 594256; fax +44 (0) 1703 594269;

e-mail nm4@soton.ac.uk

Mar. Biotechnol. 1, 533–544, 1999

© 1999 Springer-Verlag New York Inc.

the newly fertilized egg shortly after fertilization, the second

polar body, which is normally extruded after fertilization, is

retained, and as a result, the fertilized egg contains three

haploid nuclei that, on fusion, form a zygote nucleus that is

triploid (Tave, 1993). Sterility can prevent uncontrolled re-

production and is thus beneficial when the cultivation pe-

riod extends beyond sexual maturity (Boulanger, 1991).

Most importantly, it can create fishes that will not be able to

hybridize and contribute to the local gene pool if they were

to accidentally escape from pond confinement (Tave, 1993).

Sterility is induced by triploidy owing to the occurrence of

gametic incompatibility during meiosis since triploids with

three homologous chromosomes cannot align well and

there is impediment to the pairing of chromosomes during

meiosis I. This brings about difficulty in meiosis as the

chromosomes cannot split equally, resulting in uneven or

aborted separation of chromosome triplets (Myers, 1986).

As a result, gametes that contain unbalanced numbers of

chromosomes, termed aneuploids, are produced, and an-

euploid gametes usually produce abnormal and subviable

offspring that rarely survive (Benfey, 1991). Many studies

have shown that triploids not only have abnormal gonads,

but also produce fewer gametes, which are themselves usu-

ally abnormal (Nagy, 1987; Penman et al., 1987; Kim et al.,

1988; Pandian and Varadaraj, 1988; Wolters et al., 1991).

Triploids were first artificially produced in the stickle-

back fish through the application of heat shock to normally

fertilized eggs (Swarup, 1959). Since then, triploids pro-

duced by heat shock have been produced in many other

species such as channel catfish (Chrisman et al., 1983), rain-

bow trout (Quillet et al., 1988; Kim et al., 1988), common

carp (Hollabecq et al., 1988), and tilapia (Penman et al.,

1987; Don and Avtalion, 1988; Varadaraj and Pandian 1990;

Hussain et al., 1991). Apart from heat shock, other means of

producing triploid fish, such as cold shock, pressure shock,

and chemicals, have also been employed by various re-

searchers (Tave, 1993).

Tilapias are a source of cheap protein especially in un-

derdeveloped tropical and subtropical countries. Prior re-

search interest in the production of triploid tilapia has fo-

cused on trying to solve the problem of precocious maturity

and uncontrolled reproduction, which often resulted in

overpopulation of young fish in culture ponds (Bramick et

al., 1995). In recent years, our laboratory has successfully

produced transgenic tilapia with elevated growth perfor-

mance (Rahman et al., 1998; Rahman and Maclean, 1999).

Due to concern about the impact of these fish on the natu-

ral environment in the event of escape to the wild, we are

currently examining methods of inducing sterility. One of

these is by heat-shocking the fertilized transgenic eggs in

order to produce sterile transgenic triploids, although such

fish would also have to be securely contained. We are also

working on an alternative transgenic strategy to guarantee

sterility, but this may be some years away.

The benefit of induced triploidy on growth enhance-

ment is not normally evident until after the period of matu-

ration in nontransgenic diploids whereby inhibition of go-

nadal development may result in increased somatic growth

(Wolters et al., 1982). Because our transgenic tilapia are

growth enhanced at a much earlier period, we are interested

in finding out whether attainment of triploid status has any

effect on the already growth-enhanced tilapias in both non-

transgenic and transgenic adult fish as compared with nor-

mal diploid nontransgenic tilapias.

Through the combination of the techniques of chro-

mosome set manipulation (triploidy induction) and genetic

engineering (transgenic induction), we aim to produce trip-

loid transgenic tilapias that are sterile. Our research interest

in triploidy is not in triploidy per se, but in its ability to

disrupt gonad development which will thus render our

growth enhanced fish sterile.

MATERIALS AND METHODS

DNA Construct of Transgenics

The fishes used were the G3 progeny of founders of trans-

genic tilapias with DNA constructs OPAFPcsGH and

CarpbALacZ. The OPAFPcsGH is a growth hormone con-

struct containing the ocean pout antifreeze gene promoter

in addition to the 58 and 38 noncoding sequences of the

gene spliced to a chinook salmon growth hormone cDNA

(Du et al., 1992) (kindly provided by Prof. C.L. Hew,

University of Toronto, Canada). The other construct,

CarpbALacZ, is a reporter gene construct which comprises

the Carpb-actin regulatory sequences spliced to the bacte-

rial b-galactosidase gene (Alam et al., 1990). Both these

inserts were coinjected to produce the founders of the two

lines (C118 and C86) whose G3 progenies were used in this

study (Rahman et al., 1997). These G3 progenies were pro-

duced by crossing G2 hemizygous transgenic males of each

line with wild-type females. Production of transgenics and

construct details were described in Rahman et al. (1997).

Transgenic Analysis

Since these fishes coexpressed both salmon growth hor-

mone and b-galactosidase, transgenic status could be con-

534 Shaharudin Abdul Razak et al.

firmed through the detection of lacZ protein expression in

the muscle tissues of the individual tilapia adults by histo-

chemical in situ staining using X-gal (5-bromo-chloro-3-

indolyl-b-D-galactosidase) as a substrate. LacZ expression

can be easily detected as a blue precipitate at the single-cell

level, and thus it represents an in situ visual marker of the

transgene. This method was chosen because it is much less

tedious than immunohistochemical methods. Briefly, the

adult tilapias were lethally anesthesized, and the muscles

were excised and immediately fixed in freshly made fixative

(2% paraformaldehyde, 0.2% glutaraldehyde, 0.02% Non-

idet P-40, in 0.1 M sodium phosphate, pH 7.6) for 5–6

hours at 4°C. The muscle tissues were washed in phosphate-

buffered saline (PBS), pH 7.6, for 3 × 5 minutes, and then

immersed in X-gal histochemical reaction mixture (1 mg/

ml X-gal, 100 mM sodium phosphate, pH 7.6, 1.3 mM

MgCl2, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 0.02% Non-

idet P-40). Incubation was at 30°C overnight. Upon

completion of staining, tissues were washed in PBS (3 × 5

minutes), photographed, and then stored at 4°C.

Triploid Induction

Eggs were heat-shocked at 41°C, 5 minutes after fertiliza-

tion, in batches of between 200 and 250 eggs, held in a mesh

basket for 3.5 minutes duration as described by Hussain et

al. (1991). Following heat shocking, the eggs were carefully

placed in plastic conical funnels and incubated at 28°C in an

upwelling flow of water. Fish were reared in 100-L tanks at

stocking densities between 60 to 120 fish per tank. Diploid

and triploid fish were reared together, and some interactive

feeding effects may have occurred in these tanks.

Ploidy Determination

The fishes used were 8 months of age. Blood smears were

prepared by first anesthesizing the fish with 500 ppm

2-phenoxyethanol. Blood was drawn from the caudal vein

and washed in 0.2 ml of Cortland solution and 10 mM

EDTA through a disposable sterile syringe and needle (0.5 ×

16 mm). The drawn blood was smeared on to a clean mi-

croscope slide and air-dried for 30 minutes. Once dried, it

was then fixed in 100% methanol, stained in Ehrlich’s acid

hematoxylin and eosin (R.A. Lamb Ltd.), and then washed

briefly in water. The slides were then passed through an

increasing series of ethanol and finally cleared in Histoclear

(Fischer Scientific) before mounting in DPX (BDH).

Diploidy or triploidy status was determined by exam-

ining the mounted erythrocytes using an ocular micrometer

scale mounted in one of the eyepieces of a Carl-Zeiss mi-

croscope. The major axes of nuclei of individual erythro-

cytes were measured under oil immersion (1000× magnifi-

cation). It is possible that some ploidy mosaicism occurred

due to chromosome loss in some tissues, but we have never

detected this phenomenon in practice.

Gonad Histology

Gonads of each individual were collected and fixed in

freshly prepared fixative (2% paraformaldehyde/0.2% glu-

taraldehyde in 0.1 M sodium phosphate, pH 7.6). They were

then washed in PBS and stored in PBS at 4°C.

In order to ascertain whether the gonads were testis or

ovary, they were slightly squashed with a coverslip on a

microscope slide and observed under the microscope at

100× magnification.

In order to determine sterility status, the gonads were

fixed immediately after excision, embedded in paraffin wax,

sectioned to a thickness of 7 µm, and finally stained with

hematoxylin and eosin. This enabled us to observe the dif-

ferent stages of development in the gonads. Deformed, ab-

sent, or disrupted gonadal development is indicative of ste-

rility.

Gonadosomatic Index Determination

Gonadosomatic Index (GSI) was deduced according to the

formula:

~GSI! =Weight of gonad ~g!

Weight of fish ~g!× 100

Condition Factor

Condition factor (K) was deduced according to the formula:

~K! =W ~g!

@L ~cm!#3× 103

where W is the weight and L is the total length.

Design of Experiments

The fishes used in this growth trial experiment consisted of

two separate lines of growth-enhanced transgenic tilapias of

the C118 and C86 line that had previously been generated

in our laboratory (Rahman et al., 1997). These two different

lines were reared in two separate tanks as G3 progeny of a

cross between hemizygous transgenic and wild-type fish,

Growth Performance of Triploid Transgenic Tilapia 535

and as a result of the heat shock, thus are expected to consist

of control diploid nontransgenic fish, triploid nontrans-

genic fish, diploid transgenic, and triploid transgenic tila-

pias mixed in the same tank of each respective line. Trip-

loidy status was induced by heat shock. The number of

triploids and diploids present in the tank as a result of the

heat shock induction was determined at the end of the

experiment. Furthermore, since they were small, it was not

possible to determine the exact numbers of diploids and

triploids at the beginning of the experiment without sacri-

ficing them. The control tanks consisted of two separate

tanks identical in dimensions and conditions with the heat-

shock-induced transgenic and nontransgenic tilapia. One

control tank consists of the full siblings of the respective

C118 line, and the other contained the full siblings of the

C86 line. All the fishes in these control tanks were diploid

because they were not heat-shocked. Since both the experi-

mental and control fishes are the results of crosses of G2

hemizygous transgenics with wild type, the G3 progeny ob-

tained from both these crosses will theoretically be expected

to consist of 50% transgenic and 50% nontransgenics. The

fishes were fed twice a day with a commercial trout feed

pellet.

RESULTS

Ploidy Determination

At the end of 8 months, there were 117 individuals belong-

ing to the C118 line in one experimental tank and 62 indi-

viduals belonging to the C86 line in the other experimental

tank. All of them were sampled together at about the same

time. Erythrocytes of fish grown from heat-shocked fertil-

ized eggs of the C118 line revealed that 80 (68%) of the 117

were triploids. Adults sampled from the heat-shocked fer-

tilized eggs of C86 line revealed that 20 (32%) of 62 were

triploids. The rest were all diploids. Representative micro-

graphs of the erythrocytes of diploids and triploids from

both lines are presented in Figures 1a and 1b, respectively.

The triploid erythrocytes can be clearly seen to be about one

and a half times the size of diploid erythrocytes. We aimed

to use suboptimal triploid induction throughout so that a

reasonable number of diploid control fish would be avail-

able, but, in general, rates of approximately 75% triploidy

were readily obtained, and in some cases over 90% was

achieved.

Transgenic Determination

Since both transgenic lines (C118 and C86) express the

reporter gene lacZ, the easiest way to identify transgenic

individuals is by X-gal staining of muscle tissues of each

individual fish under analysis. After X-gal staining of

muscle tissues, the expressing muscle cells showed a blue

color that can be visualized by the naked eye. Both lines can

be distinguished from each other by the different level of

b-galactosidase expression exhibited between these two

lines. A higher level of b-galactosidase expression was ob-

served in the C118 line (Figure 2a) and lower expression in

the C86 line (Figure 2b). The two lines both carry the trans-

genes at a single chromosomal locus within a concatamer,

the copy number estimate being 18 for C118 and 12 for

C86. There is slight detectable variation in the level of lacZ

expression between individuals of the same line but not

enough to reveal an overlap between the lines. As expected,

the nontransgenics did not show any expression (Figure 2c).

Figure 1. Effect of heat-shock-induced triploidy on erythrocyte

size in (a) diploid (1000× magnification) and (b) triploid (1000×

magnification) tilapia.

536 Shaharudin Abdul Razak et al.

As shown by Rahman et al. (1997, 1998), we know that

growth enhancement and lacZ expression are inherited to-

gether because of close chromosomal linkage. No problems

are apparent in these lines as a result of their ubiquitously

expressing lacZ. Figure 2b suggests that many muscle fibers

in this line are not expressing lacZ, and we are following up

this observation with further study.

Body Weight

The weight distribution of nontransgenic diploids and trip-

loids and transgenic diploids and triploids from the C118

line is shown in Figure 3. Difference between means was

statistically analyzed by the Student’s t test (mean ± SEM)

(Table 1). Transgenic diploids showed the most superior

weight (121.8 ± 12.6 g) followed by transgenic triploids

(71.1 ± 5.0 g). Transgenic triploids outperformed nontrans-

genic diploids (39.2 ± 4.5 g) by 1.8 times (p < .001). The

mean weight of transgenic diploids was almost twice that of

transgenic triploids. Among the nontransgenics, the dip-

loids were superior in mean weight to triploids by about 1.5

times. Whether diploids or triploids, transgenic fish always

outweighed nontransgenics. Among the diploids, when

comparing transgenic diploids with nontransgenic diploids,

transgenic diploids were superior in mean weight by 3.2

times (p < .001). When comparing transgenic diploids with

nontransgenic triploids, transgenic diploids were much

more superior in weight to nontransgenic triploids by al-

most 5 times (p < .001). In this C118 line, it was observed

that the transgenics were superior to nontransgenics in

mean weight, and within a given status (either nontrans-

genic or transgenic), diploids outperformed triploids.

Weight distribution of diploid nontransgenic and dip-

loid and triploid transgenic tilapia from line C86 is shown

in Figure 4. There were 21 females and 41 males in this C86

population. The most surprising observation in this line is

that we could not find a single triploid nontransgenic pres-

ent in the tank. Among the transgenics, the diploids (174.9

± 17.6 g) were superior in mean weight to triploids (136.1

± 9.72 g) by almost 1.3 times (p < .001). Whether triploids

or diploids, the transgenics outperformed the nontransgen-

ics. Among the diploids, the transgenics performed much

better than nontransgenics (39.9 ± 5.6 g) by 4.4 times (p <

.001). There were no samples present in the triploid non-

transgenic group, and thus it was not possible to compare

the performance between nontransgenic diploids and trip-

loids in this C86 line.

Condition Factor

Condition factor (K) for the C118 line indicated that the

transgenics had significantly lower values than the non-

transgenics. This is evident in the transgenic diploids (29.19

± 0.42) and transgenic triploids (30.13 ± 0.30) when com-

pared to the control nontransgenic diploids (32.28 ± 0.54)

(p < .001) (Table 1). Both are also significantly different

from the nontransgenic triploids (32.28 ± 0.53) (p < .01).

However, within the transgenics, there was no significant

difference between the diploids and triploids (p > .05). This

lower K value indicates that within a given weight, the

transgenics are longer than the nontransgenics.

Figure 2. X-gal staining of muscle tissues from (a) positive C118 line (10× magnification), (b) positive C86 line (10× magnification), and

(c) negative nontransgenic tilapia (10× magnification).

Growth Performance of Triploid Transgenic Tilapia 537

In the C86 line, the only significant difference was be-

tween the transgenic diploids (30.26 ± 0.55) and the non-

transgenic diploids (32.88 ± 0.55) (p < .01). As observed in

the C118 line, there was no significant difference in condi-

tion factor between the transgenic diploids and triploids (p

> .05). There were no individuals belonging to the non-

transgenic triploids in this sample. Again, as observed in the

C118 line, the lower K values indicated that within a given

weight, the transgenics were longer than the control non-

transgenics.

Gonad Histology

Initially, gonads were identified by the naked eye, but this

was later found to be inaccurate. Hence, to ensure accuracy,

all gonads were observed histologically by paraffin section-

ing at 7 µm thickness followed by staining with hematoxylin

and eosin. Gonads were identified by observing the follow-

ing criteria: in males, the presence of spermatogonia, sper-

matocytes, or spermatozoa; and in females, the presence of

oogonia or oocytes.

The results of histologic examination of ovaries showed

that in both C118 and C86 lines, ovaries of female control

nontransgenic diploids were packed with developing oo-

cytes in various stages of development (Figure 5a). How-

ever, ovaries from triploid nontransgenic and transgenics

were small and undeveloped (Figure 5b). They contained

only oogonia, and if oocytes were present, they were usually

very few in number and of very small size or in a deformed

state.

The testes of triploids of both C118 and C86 lines were

not as abnormal as the ovaries, but it seemed that the trip-

loids did not possess as much spermatozoa as their diploid

counterparts did. Although most triploid testes appeared

reduced in size with very few spermatogonia (Figure 6b),

some still possessed spermatozoa (Figure 6c). In diploid

testes, spermatogonia completing mitotic division were

readily observed (Figure 6a).

Gonadosomatic Index

In the C118 line, the mean gonadosomatic index (GSI) for

triploid ovaries was significantly smaller both in the trans-

genics and the nontransgenics. The triploids had a very

significantly lower GSI than the diploids (p < .001). This

indicated that the gonad development was suppressed in the

Figure 3. Weight distribution of control nontransgenic diploid

[NT (2N)], nontransgenic triploid [NT (3N)], transgenic diploid

[T (2N)], and transgenic triploid [T (3N)] groups of tilapia from

C118 line of approximately 8 months of age arranged in ascending

order of weight in each group; n = number of individuals.

538 Shaharudin Abdul Razak et al.

Table 1. Mean Weight (g), Gonadosomatic Index (GSI) of Females and Males, and Condition Factor (K) of Control Nontransgenic (NT) Diploid (2N), Nontransgenic

(NT) Triploid (3N), Transgenic (T) Diploid (2N), and Transgenic (T) Triploid (3N) of Tilapia (Oreochromis niloticus) of approximately 8 Months of Age from C118 and

C86 Lines†

Transgenic

lines

Percentage

transgenic

Weight (g) GSI females GSI males Condition factor (K)

NT T NT T NT T NT T

2N 3N 2N 3N 2N 3N 2N 3N 2N 3N 2N 3N 2N 3N 2N 3N

C118 38 n = 26 n = 46 n = 13 n = 32 n = 8 n = 22 n = 6 n = 13 n = 18 n = 24 n = 7 n = 19 n = 26 n = 46 n = 13 n = 32

39.2

± 4.5

25.9

± 2.9

121.8

± 12.6

71.1

± 5.0

0.895

± 0.3

0.0993

± 0.0146

0.0373

± 0.0118

0.0247

± 0.003

0.0736

± 0.0013

0.0984

± 0.0159

0.0326

± 0.0024

0.0736

± 0.0127

32.278

± 0.537

32.278

± 0.531

29.186

± 0.419

30.131

± 0.300

** *** *** *** * *** ** NS * NS *** ***| | | | | | | | | | | | | | | |

*** *** *** NS *** NS * *| | | | | | | | | | | |

*** *** *** NS| | | | | | | |

C86 56 n = 27 NA n = 15 n = 20 n = 11 NA n = 7 n = 20 n = 16 NA n = 8 NA n = 27 NA n = 15 n = 20

39.851

± 5.57

174.93

± 17.6

136.10

± 9.72

0.440

± 0.174

0.0662

± 0.0076

0.0129

± 0.0028

0.113

± 0.0419

0.0558

± 0.0073

32.877

± 0.554

30.258

± 0.553

31.916

± 0.617

*** *** NS ** NS ** NS| | | | | | | | | | |

* *** NS| | | | | |

†All means of each status are connected by the same horizontal line in each physiological condition (with different asterisks indicating different statistical significance levels) and are compared

with the group situated at the extreme left of each line (without asterisks). Statistical significance was measured at *p < .05; **p < .01; ***p < .001, using Student’s t test. NS, not significant;

NA, not available, n, number of individuals; NT, nontransgenic; T, transgenic; 2N, diploid; 3N, triploid.

Grow

thP

erforman

ceof

Triploid

Tran

sgenic

Tilapia

539

triploids. Some individuals had only single previtellogenic

oocyte, and most had only connective tissue cells. Triploid

ovaries, owing to their smaller size, were easy to distinguish

from diploid ovaries, as many were very stringy in appear-

ance and seemed fragile. GSI values for nontransgenic

(0.0993 ± 0.0146) and transgenic triploids (0.0247 ±

0.0030) were very significantly less than for control non-

transgenic diploids (0.8954 ± 0.3) (p < .001) (Table 1).

This similar GSI condition was also observed in the

testes of this line. The nontransgenic triploid testes were

significantly smaller than those of transgenic diploids (p <

.01). The transgenic triploid testes were significantly smaller

than those of the nontransgenic diploids (p < .01). How-

ever, there was no significant difference between the trans-

genic diploids and the control nontransgenic diploids (p >

.05). There was also no significant difference between the

transgenic triploids and the nontransgenic triploids. The

transgenic diploid have significantly higher GSI than both

the nontransgenic triploids and the transgenic triploids (p <

.001). Generally few spermatozoa were observed in triploid

testes, although they were not completely absent and thus

could still be functional; therefore, the fish may not be

completely sterile (Figure 6c). However, because of the in-

creased size of triploid sperm, fertilization may be reduced

or absent due to the narrow dimensions of the egg micro-

pyle.

In the C86 line, there were no nontransgenic triploids

present in the sample. There were also no males present in

the transgenic triploid samples. There was no significant

difference in mean GSI between the nontransgenic diploids

and transgenic diploid females (p > .05). GSI of females of

the transgenic triploids was significantly lower when com-

pared with the nontransgenic diploids (p < .01). Since non-

transgenic triploids were not present in the sample, it was

not possible to compare the mean GSI with the nontrans-

genic diploids. However, between the transgenics, the trip-

loids (0.0129 ± 0.0028) were significantly lower than the

diploids (0.0662 ± 0.0076) (p < .001).

Regarding the males of this C86 line, because no males

were present in the transgenic triploids, and no nontrans-

genic triploids were found to be present in the samples, the

only comparison that can be made was between transgenic

diploid (0.0558 ± 0.0073) and nontransgenic diploids

Figure 4. Weight distribution of control nontransgenic diploid

[NT (2N)], transgenic diploid [T (2N)], and transgenic triploid [T

(3N)] groups of tilapia from C86 line of approximately 8 months

of age arranged in ascending order weight in each group; n =

number of individuals.

Figure 5. Histologic section (7 µm) of ovary from (a) diploid

(500× magnification) and (b) triploid (600× magnification) tila-

pia.

540 Shaharudin Abdul Razak et al.

(0.113 ± 0.0419). There was no significant difference be-

tween the mean GSI (p > .05) (Table 1).

DISCUSSION

To the best of our knowledge, this is the first report of an

attempt to determine the effect of induced triploidy on

growth-enhanced transgenic fish. Our results show that the

transgenic diploids weigh more than the nontransgenic dip-

loids, nontransgenic triploids, and transgenic triploids. In

the C118 line, the transgenic diploids were 3.2 times heavier

than the nontransgenic diploids. Controlled growth trials of

these lines have already been published (Rahman et al.,

1998), and data from more extensive growth trials are in

preparation for publication. In the C86 line, the transgenic

diploids were superior by 4.4 times to the nontransgenic

diploids. This result is in accordance with the observation of

Rahman et al. (1998) in both these lines. Our results clearly

indicate that in both lines, the triploid nontransgenics are in

fact inferior to the diploid nontransgenic tilapias. Among

the transgenics, although the triploids were inferior in their

growth to diploids, they were still superior to either diploid

or triploid nontransgenics.

Condition factor (K) for both lines showed no signifi-

cant differences between diploids and triploids within a

given nontransgenic or transgenic strain. Regardless of

whether they are diploid or triploid, the transgenics had

lower K values than the nontransgenics. Our result is partly

in agreement with Benfey and Sutterlin (1984), who re-

ported that triploid Atlantic salmon weighed less than their

diploid counterpart; however, it is in disagreement with

their finding that triploids were longer than diploids.

Gonad histology indicated that the triploids are most

probably sterile owing to disruption in gonad development.

In the ovaries of triploids, we observed that most individu-

als had no oocytes at all, although occasionally we detected

one or two abnormal oocytes. This finding is similar to

those of Okada (1985) and Nakamura et al. (1987) in trip-

loid rainbow trout. Okada (1985) suggested that germ cells

developed up to the karyosome stage, but unlike diploids,

which enter meiosis with concomitant oocyte growth, they

then regressed. Nakamura et al. (1987) observed many syn-

aptonemal oocytes in the nuclei of many reproductive cells,

which were confirmed to be oocytes in the stage preceding

the first meiosis. Both these observations suggest that the

sterility of triploids arises because the chromosomes cannot

separate after pairing in the first maturation cleavage. Off-

spring of triploids, when they do occur, are usually abnor-

mal and die when still in the embryonic stage (Nagy, 1987;

Penman et al., 1987). Although Lincoln (1981) was able to

induce triploid plaice to produce offspring, none were able

to hatch successfully. This was shown by Nagy (1987), who

mated sex-reversed triploid gynogenetic silver carp males

with diploid common carp females and produced only two

individuals that survived past first feeding. Testes of trip-

Figure 6. Histologic section (7 µm) of testis from (a) diploid

tilapia (500× magnification), (b) triploid (600× magnification)

tilapia, and (c) spermatozoa (1000× magnification) of triploid

tilapia.

Growth Performance of Triploid Transgenic Tilapia 541

loids in both lines (C118 and C86) looked similar in mor-

phology to diploids without any sign of gross deformity but

were significantly smaller. Other workers made the same

observation in other triploid fishes (Lincoln and Scott,

1984; Allen et al., 1986; Ueno et al., 1986; van Eenennaam

et al., 1990). On dissection, triploid testes were found to

possess seminal fluid but very little spermatozoa and could

therefore still bring a certain degree of fertility to the trip-

loid males. This is similar to the observation of Hussain et

al. (1995) in this same O. niloticus species. In male Tilapia

macrocephala, Aronson (1995) found that 7.5% of testicular

tissue was sufficient to maintain normal secondary sexual

characters. This condition is also reflected in the GSI, which

generally showed lower values in triploids than in diploids.

This indicated that gonad development was suppressed in

the triploid females and therefore could have diverted the

energy normally invested in gonad development to somatic

growth. However, our observation is in contrast to the find-

ings of other workers who obtained high GSI values in

triploid female tilapias (Penman et al., 1987; Bramick et al.,

1995; Hussain et al., 1995), rainbow trout (Lincoln and Bye,

1987), and Pacific salmon (Benfey et al., 1989). It may be

that the fish we have used are not old enough to reveal the

ultimate GSI value.

There are many reports that triploids do not grow well

when cultured in competition with diploids (Penman et al.,

1987; Thorgaard, 1992; Galbreath et al., 1994). This is in

agreement with the results obtained from this study reveal-

ing that within the same status, whether nontransgenics or

transgenics, the triploids were found to be inferior to the

diploids. This is shown in the C118 line. It is even remark-

ably shown in the C86 line, in which we could not find even

a single nontransgenic triploid present in the sample. It is

possible that the aggressive nature of the transgenics in

competition for food and the establishment of hierarchy

dominance by the transgenics would make the triploids very

inferior and thus make them the recipient of agonistic ac-

tions (Abbott and Dill, 1985). In this study, the complete

absence of the nontransgenic triploid fish in the sample of

the C86 line could be the outcome of a combination of the

inferiority of the nontransgenic triploid and the aggressive-

ness of the transgenics. However, the inferiority of the trip-

loids could be overcome by being transgenic, as shown in

this study in the C86 line in which it outperformed the

nontransgenic diploid controls. This result is similar to that

observed in salmonids, in which size can influence rank,

and larger individuals are usually more successful in com-

peting for a resource (Abbott et al., 1985).

Increase in DNA content resulting from triploidy leads

to an increase in both nuclear and cellular volume in a wide

range of tissues, but cell numbers are reduced to maintain

normal organ and body size (Swarup, 1959). Visual acuity

and learning ability could be impaired due to decrease in

cell numbers observed in retinal and brain cells of triploids

(Small and Benfey, 1987). Triploid fish have also been

shown to be less sensitive to sound and light than diploids

(Aliah et al., 1990). These observations indicate that trip-

loids could face behavioral or learning disadvantages rela-

tive to their diploid counterparts, which should be of con-

cern in aquaculture because it could lead to decreased fit-

ness especially under suboptimal conditions of rearing

(Quillett and Gaignon, 1990). Our findings that the growth-

enhanced transgenic triploids did not encounter any nega-

tive behavioral or learning disadvantages suggest that they

were not affected by the problems associated with cell size.

If they were affected, they would presumably be in the same

position as the nontransgenic triploids, which were annihi-

lated in the C86 line (and, incidently, in terms of weight

became the most inferior group in the C118 line). A pos-

sible explanation for this observation is that, because the

growth-enhanced transgenic triploids were much bigger

than the nontransgenic triploids, it follows that they also

possess a bigger skull than the nontransgenic triploids. This

will enable them to accommodate more cells due to the

much bigger volume available to them. The eventual out-

come will be a ratio of number of brain cells to overall brain

volume available that would be similar to that of the non-

transgenic diploids, and since nothing is compromised, they

thus did not suffer any adverse consequences. In order to

maintain normal organ and body size, the smaller brain

volume available to the nontransgenic triploids will accom-

modate a reduced number of cells compared to the triploid

transgenics, and this could lead to impairment of visual

acuity and learning ability.

Penman et al. (1987) concluded that triploids of Oreo-

chromis species produced by heat shocks would not be of

benefit to aquaculture because of their reduced growth rate.

This study also confirms their findings that the triploids are

inferior in weight to diploids if they are both nontransgen-

ics. The comparative performance of triploid and diploid

fish varies from species to species, no doubt as a result of the

variation in reproductive activity during the life period.

However, with the use of growth-enhanced transgenic tila-

pias, a totally different scenario emerges whereby even the

triploid transgenics outperform both diploids and triploid

nontransgenics. Although the best growth performance in

542 Shaharudin Abdul Razak et al.

this study was shown by the diploid growth-enhanced

transgenics, this will not be a good choice for aquaculture as

the gonads of the females are viable, and there is clear

concern that they could breed with the wild population and

that the transgene will introgress into the gene pool of the

wild. This study has shown that a possible option will be to

come to a compromise solution on growth enhancement by

sterility through the acquirement of triploidy status. This

will still make it very much superior to the normal wild-

type tilapias.

Triploid fish are commonly functionally sterile (Lin-

coln and Scott, 1984; Hussain et al., 1995). However, there

is still a distinct possibility that the transgenic triploids

could create problems as far as reproductive viability is

concerned owing to the presence of a small amount of

spermatozoa in the testes. Hence, the culture of males will

be highly risky to the environment in its ability to fertilize

the wild population if it managed to escape from pond

confinement. However, as mentioned earlier, the effective-

ness of triploid sperm in fertilization may be impaired due

to the increased size of the sperm head. A possible option to

undertake in the future will be to produce all-female

growth-enhanced triploid transgenic tilapias. As shown in

this study, the female gonads of the triploids are certainly

sterile as most of them do not contain even a single oocyte,

and those that contain oocytes are usually too few in num-

ber and are usually deformed. However, rigorous breeding

studies would be necessary prior to commercial use to en-

sure that no gene flow occurred.

Since we did not wish to have high rates of triploid

induction in these experiments because we used the diploid

as controls, 68% triploidy in the C118 line and 32% trip-

loidy in the C86 line after heat-shock induction was used. In

another batch, we obtained 90% triploidy in the C118 line.

In order for heat induction to be used routinely to create

triploids, a 100% yield would be the ideal target. Yields

obtained by other researchers are quite variable, but some

did succeed in obtaining 100% triploidy yield in their in-

ductions (Hussain et al., 1991). If proper timing of heat

induction can be effectively optimized, it may be possible to

achieve this objective.

Our inability to detect any triploid nontransgenic fish

in the C86 line is puzzling; it may simply be an anomaly of

the small sample size.

Although triploid transgenics are growth enhanced and

sterile, the leakiness of the procedure means that it will still

be necessary to attempt to gain 100% sterility through a

transgenic manipulation before it will be entirely safe to

release growth-enhanced transgenic fish for general use in

aquaculture. Our laboratory is currently involved in this

line of research.

ACKNOWLEDGMENTS

We are very grateful to Prof. C.L. Hew (University of To-

ronto, Canada) for providing us with the growth hormone

gene constructs. Thanks are also due to Dr. Arati Iyengar

for reading the manuscript. S.A.R. was funded by the Uni-

versity of Malaya, Malaysia, and G.L.H. by the Korean Sci-

ence and Engineering Foundation. The transgenic fish used

in this work were from strains produced with the support of

the Department for International Development (DFID)

Fish Genetics Research Programme.

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