RESEARCH ARTICLE

Ovarian Development of a River

Catfish Hemibagrus nemurus
(Valenciennes, 1840) in Captivity
FATIMAT ADENIKE ADEBIYI1, SITI SHAPOR SIRAJ1,
SHARR AZNI HARMIN2, AND ANNIE CHRISTIANUS1
1
Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, Selangor, Malaysia
2
Department of Aquatic Biotechnology, Faculty of Science and Technology, Universiti Industri
Selangor, Selangor, Malaysia

ABSTRACT Hemibagrus nemurus is a riverine catfish with high economic and nutritive values. Investigations
on ovarian development of this fish were carried out to determine the mode of ovarian
development and describe the oocyte developmental stages. Histological studies were done on
ovaries using light microscopy and scanning electron microscopy. Fish were sampled monthly for a
period of six months (August 2009 to January 2010). The mean oocyte diameter (OD) ranged from
8717161.41 mm to 1,167726.77 mm and the highest OD was in November. Oocyte size–frequency
distribution showed a polymodal distribution. The mean gonadosomatic index (GSI) ranged from
1.1470.87% to 7.0671.40% and highest GSI was in November. The ovaries exhibited three
phases of oocyte growth, which were primary growth, secondary growth and maturation phases.
Based on histological criteria, the oocyte developmental stages were divided into seven stages as
chromatin nucleolar, early perinucleolar, late perinucleolar, cortical alveolar, vitellogenesis, mature
oocyte and germinal vesicle migration stages. All the seven stages of oocyte development were
observed in the ovaries. Oogonia were always present throughout the developmental stages. The
ovaries had more than two stages of oocyte development. This is the first report on the mode of
ovarian development of H. nemurus. These findings indicated that H. nemurus has asynchronous
mode of ovarian development and is capable of spawning several times in a year under favourable
conditions. J. Exp. Zool. 315:536–543, 2011. & 2011 Wiley Periodicals, Inc.

J. Exp. Zool. How to cite this article: Adebiyi FA, Siraj SS, Harmin SA, Christianus A. 2011. Ovarian development of
315:536–543, 2011 a river catfish Hemibagrus nemurus (Valenciennes, 1840) in captivity. J. Exp. Zool. 315:536–543.

The knowledge of reproductive biology of fish is very vital for

successful fish culture. The gonads play an important role in
reproduction. Hence, for fish to reproduce, the gonads need to be
well developed and matured. Oogenesis and spermatogenesis are
Grant Sponsor: Ministry of Agriculture, Malaysia; Grant number: 5487718;
the two reproductive processes that occur in female and male fish
Grant Sponsor: Internal Funding from Universiti Putra Malaysia.
during the reproductive cycle, leading to the production of mature
Abbreviation: ANOVA, Analysis of variance; GSI, Gonadosomatic index; OD,
oocytes and spermatocytes. During the process of oogenesis,
Oocyte diameter; OO, Oogonia; I, Chromatin nucleolar oocyte; II, Early
the ovary undergoes different developmental stages, which include perinucleolar oocyte; III, Late perinucleolar oocyte; IV, Cortical alveolar
oocyte growth, development and maturation. Ovarian development oocyte; V, Vitellogenic oocyte; VI, Mature oocyte; VII, Germinal vesicle
occurs in three phases which are the primary growth phase, migration oocyte.
Correspondence to: Siti Shapor Siraj, Department of Aquaculture, Faculty
secondary growth phase and maturation phase (Coward and
of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,
Bromage, ’98; Cek et al., 2001). During these phases the oocytes
Malaysia. E-mail: shaporsiraj@yahoo.com; shapor@putra.upm.edu.my
undergo a series of development. The primary growth phase is Received 5 March 2011; Revised 28 June 2011; Accepted 20 July 2011
usually characterized by previtellogenic oocytes, while the Published online 6 September 2011 in Wiley Online Library (wileyonline
secondary growth phase is characterized by vitellogenic oocytes library.com). DOI: 10.1002/jez.702

& 2011 WILEY PERIODICALS, INC.

OVARIAN DEVELOPMENT OF Hemibagrus nemurus 537

and the maturation phase is characterized by advanced vitellogenic Selangor, Malaysia. After disinfection with potassium permanga-
oocytes undergoing germinal vesicle migration (Estay et al., ’98). nate solution, the fish were kept in 40 tonnes flow through
There are three types of ovarian development (Selman and concrete rectangular tanks under natural photoperiod to acclima-
Wallace, ’89), such as asynchronous, different stages of oocyte tization for two months. Feeding to satiation was done once daily
development are found in the ovary; group synchronous, at least around 10:00 a.m. with artificial pelleted feed (Cargill) with a
two population of different stages of oocyte development are dietary composition of 38–40% crude protein, 13% moisture, 6%
found in the ovary or synchronous, all the oocytes develop fiber and 3% crude fat. The feeding rate was 3% body weight of
concurrently. Gonadosomatic index (GSI) is an important fish. Thirty females out of the stock were used for this study.
indicator of reproductive activity and is used for the determina- Five female fish were randomly sampled every month and
tion of gonadal maturation (Hojo et al., 2004). transferred to one tonne circular fiber glass tank for a period of
Among the bagrid fish, Hemibagrus nemurus is one of the six months starting from August 2009 to January 2010. All fish
tropical freshwater catfish which is native to Asian waters and were anaesthetized with clove oil (0.1 ml/l) before handling. Body
can be found in countries such as Malaysia, Indonesia, Cambodia, weight of fish were measured using a spring balance, gonad
Laos, Thailand and Vietnam (Rainboth, ’96). It dwells in ponds, weight was measured using a digital scale balance with 0.001
swamps, streams, lakes and rivers (Inger and Chin, ’62). It is sensitivity and body lengths (standard and total lengths) were
economically valued for its importance in fisheries and aqua- measured using a one-meter measuring board. Water quality
culture. Its food value which includes high protein and omega-3 parameters such as temperature, dissolved oxygen and pH of the
polyunsaturated fatty acid and low cholesterol also makes it a tank were monitored and maintained at 27.0–27.51C,
good fish species for aquaculture (Mesomya et al., 2002). 7.72–8.02 mg/l and 7.74–7.80, respectively.
Three quarter of the world human population is dependent on The fish were decapitated and dissected to remove the ovaries.
fish for primary source of protein. Hence, efforts are being made to The GSI was calculated as ovary weight divided by body weight
replenish the wild stocks and improve the aquaculture industry of multiplied by 100. The oocyte diameter (OD) was measured with
economically important fish species like H. nemurus. Breeding ocular micrometer (Nikon SMZ 045). One hundred oocytes were
programmes could be done to achieve this aim. Studies on its sampled from each female fish. The oocytes were collected from
reproductive biology will enhance our knowledge about its the midlobe of the ovary and preserved in 0.6% saline and 1%
reproduction and would enable us to have a feasible and achievable formalin solution and measured.
programme for its culture and management. Although the
commercial and intensive culture of H. nemurus is not well Gonad Histology
established, interests in its aquaculture industry is emergent (Khan For histological studies, samples of ovarian tissues were obtained
et al., ’90). from the midlobe of the ovary and preserved in bouin’s solution for
In the past researches, studies on the aquaculture of 24 hr. The tissues were later transferred to 70% ethanol and
H. nemurus have been done significantly in areas such as processed using the routine histological methods. Tissue sections
nutrition, genetics, growth and cryobiology (Khan et al., ’91; were cut at 4 mm. Cut sections were mounted on glass slides and
Hoque et al., ’98; Eguia et al., 2000; Ng et al., 2000, 2001; stained using Haematoxylin and Eosin staining technique. After
Muchlisin et al., 2006; Hoh et al., 2008; Keong et al., 2008; staining, the slides were viewed with a light microscope (Leica DFC
Muchlisin and Siti Azizah, 2009). Few works have been reported 295, Leica Microsystems, Queenstown, Singapore). The oocyte
on the reproductive biology of this fish (Khan et al., ’90; development stages were described following the histological
Christianus et al., ’98, ’99; Muchlisin and Siti Azizah, 2009) and criteria of Coward and Bromage (’98). Oocyte size was estimated
some information on its reproductive biology are still unknown. on at least 30 oocytes at each developmental stage according to the
Information on the ovarian development of H. nemurus is method of Estay et al. (’98). In order to study the surface structure of
scarce. Ovarian development studies would provide useful informa- the ovaries, samples were prepared for scanning electron micro-
tion on its spawning pattern and strategy, which is very important scopy. The ovarian tissues were cut into 1 cm3 sizes and fixed in 4%
for sustaining the aquaculture of H. nemurus in the country. Thus, glutaraldehyde for 12–24 hr, at 41C. A 0.1 M sodium cacodylate
this study highlights the study on the mode of ovarian development buffer was used to wash the samples, and then post fixed in 1%
and the oocyte developmental stages of H. nemurus. osmium tetraoxide for 2 hr at 41C. Thereafter, 0.1 M sodium
cacodylate buffer was used to wash the samples. The samples were
later dehydrated using series of acetone (35, 50, 75, 95 and 100%).
MATERIALS AND METHODS
Samples were transferred into specimen basket and put into a
Experimental Fish, Maintenance and Experimental Design critical point dryer (Baltec CPD, Baltec Corporations, Canonsburg,
Adult female H. nemurus were collected from Pahang river, PA) for 30 min. After the critical point drying of samples, samples
Pahang, Malaysia. The fish were transported to the Universiti were mounted on stubs and coated with gold using the sputter
Putra Malaysia Aquaculture Research Station in Puchong, coater (Baltec SC 030, Baltec Corporations, Canonsburg, PA). The

J. Exp. Zool.
538 ADEBIYI ET AL.

samples were then viewed with a scanning electron microscope

(ESEM Phillips XL30, FEI Company, Hillsboro, OR).

Statistical Analysis
Computer statistical analysis software (SAS 9.2) was used. Data
were expressed as mean7standard error of mean. One way
analysis of variance (ANOVA) followed by Duncan’s New
Multiple Range Test was used for mean variation. The level of
significance selected was Po0.05.

RESULTS
Ovarian Morphology, OD and GSI
Like most teleosts, the ovary of H. nemurus was an elongated
tubular sac-like structure, which contained oocytes. The ovary
had two lobes and was light brown in color. It was attached to the
abdomen by the dorsal mesentery and extended posteriorly into
Figure 1. Oocyte diameter (OD) of H. nemurus. the oviduct. Mature ovaries were large, robust and completely
filled with oocytes while immature ovaries were small and thin.
OD, which is a measurement of oocyte growth, ranged from
500 mm in immature oocyte to 1,450 mm in mature oocyte. The
mean OD ranged from 8717161.41 mm to 1,167726.77 mm. OD
was significantly different (Po0.05) during the sampling months
and the highest OD was observed in November (Fig. 1).
The oocyte size–frequency distribution (Fig. 2) showed a
polymodal distribution.
The GSI ranged from 1.1470.87 to 7.0671.40. The GSI was
significantly different during the sampling months (Po0.05). The
highest gonadosomatic was observed in November (Fig. 3).

Figure 3. Gonadosomatic index (GSI) of H. nemurus. OO, oogonia;

I, chromatin nucleolar oocyte; II, early perinucleolar oocyte; III, late
perinucleolar oocyte; IV, cortical alveolar oocyte; V, vitellogenic
Figure 2. Oocyte size–frequency distribution of H. nemurus. oocyte; VI, mature oocyte). Scale bar 5 200 mm.

J. Exp. Zool.
OVARIAN DEVELOPMENT OF Hemibagrus nemurus 539

Table 1 shows the sampling months, body weight, gonad weight The oocyte size–frequency distribution showed a polymodal
and GSI of H. nemurus. frequency distribution in almost all the months. This was in
conformity with the work of Khan et al. (’90). A similar oocyte
Gonad Histology size–frequency distribution was observed in oxleyan pygmy
The ovary in the primary growth phase had immature oocytes of perch (Nannoperca oxleyana) (Knight et al., 2007). This indicates
chromatin nucleolar, early perinucleolar and late perinucleolar asynchronous spawning pattern. More than two oocyte stages
stages. Cortical alveolar and vitellogenesis stage oocytes were dominated the ovary, which implied that a continuous breeding
observed in ovaries at the secondary growth phase. Mature season is apparent, where mature oocytes are ovulated during
oocytes were found in the ovaries at maturation growth phase. favorable conditions. Breeding season of H. nemurus is
Mature ovaries had oocytes in mature and germinal vesicle
migration stages. It was observed that the ovaries had oocytes of
more than two stages (Figs. 4A and B) and some ovaries had
nearly all the stages of oocyte development present (Fig. 4C).

Description of Oocyte Developmental Stages

The oocyte developmental stages were divided into seven stages
as follows: stage I, chromatin nucleolar oocyte; stage II, early
perinucleolar oocyte; stage III, late perinucleolar oocyte; stage IV,
cortical alveolar oocyte; stage V, Vitellogenic oocyte; stage VI,
mature oocyte and stage VII, germinal vesicle migration oocyte
stages. Table 2 shows the description of the different stages of
oocyte development based on histological criteria (Fig. 5).
Scanning electron microscopy revealed that the oocyte had a
smooth surface (Fig. 6A). Electron micrographs of immature and
mature ovary also confirm the presence of different oocyte stages
in the ovary (Fig. 6B and C).

DISCUSSION
The morphology of the ovary of H. nemurus was similar to most
teleost fish (Ravaglia and Maggese, 2002; Van Der Molen and
Matallanas, 2004; Abascal and Medina, 2005; Knight et al., 2007;
Solomon and Ramnarine, 2007; Koc et al., 2008; Chelemal et al.,
2009).
A high OD, which was observed in November, implied that the
Figure 4. Cross section of ovary of H. nemurus. (A) Immature ovary
oocyte reached maturity in this month and ovulation is likely to
showing more than two stages of oocyte development. (B) Mature
occur. Therefore, the fish would spawn around this month under
ovary showing more than two stages of oocyte development.
suitable conditions. According to Khan et al. (’90), OD peaked in
(C) Ovary with almost all the stages of oocyte development.
April and September, indicating the spawning period.

Table 1. Morphometric measurements and gonadosomatic index.

Sampling Total length Standard length Body weight Gonad weight GSI
months (cm)7SEM (cm)7SEM (g)7SEM (g)7SEM (%)7SEM
August 38.571.38 31.9271.10 572753.89 11.7474.29 2.0670.87
September 40.3471.22 33.4871.05 670766.93 23.4275.49 3.4370.84
October 35.5470.93 30.3270.80 500736.33 21.4179.37 4.4772.02
November 42.271.29 35.6471.04 888782.85 65.27717.22 7.0671.40
December 40.471.44 32.870.46 593744.43 6.7370.43 1.1470.02
January 36.5271.25 30.8271.07 544747.92 18.5578.93 3.3371.58

Data are mean7SEM.

J. Exp. Zool.
540 ADEBIYI ET AL.

Table 2. Description of oocyte developmental stages.

Oocyte
Growth diameter
phases Stages (mm) Features
Primary I 30–90 The oocyte had cytoplasm, a central nucleus with a single nucleolus. Chromatin threads were observed in
the nucleus (Fig. 5A)
II 55–145 The oocyte still had cytoplasm and central nucleus. The nucleus had many nucleoli. These were provitelline
nucleoli. Chromatin threads were present in the nucleus. The oocyte membrane was surrounded by
developing follicular cells and the oocyte appeared to be purple in colour (Fig. 5B)
III 140–400 The oocyte cytoplasm was narrow and the nucleus was large, contained chromatin threads and still
remained in the central position. The number of nucleoli in the nucleus increased and they appeared near
the nuclear membrane forming a clear ring on the nuclear membrane. The follicular layer surrounding
the oocyte was evident at this stage and the zona radiata can be differentiated from the follicular
epithelium. The oocyte increased in size at this stage (Fig. 5C)
Secondary IV 250–500 The cytoplasm at this stage appeared to be light pink in color. Cortical alveoli were observed around the
oocyte cortex. Yolk granules and yolk vesicles were present in the cytoplasm. The yolk vesicles were
observed to be close to the oocyte periphery. The nucleus was smaller compared to the earlier stages and
chromatin threads still occurred in the nucleus. The nuclear membrane began to degenerate. The zona
radiata layer was very evident and follicular epithelium was more developed (Fig. 5D)
V 400–700 The oocyte began to undergo vitellogenesis at this stage. The oocyte appeared to be pinkish red. The yolk
vesicle and granules were observed in the whole cytoplasm area. The nuclear membrane was completely
degenerated. Zona radiata and follicular layers were now well developed and distinct. There was an
increase in the size of oocyte (Fig. 5E)
Maturation VI 450–1,000 The oocyte was pinkish in color and bigger in size. The yolk granules observed in the cytoplasm completely
filled up the cytoplasm. The zona radiata and follicular layer were fully developed and were differentiated
as distinct layers (Fig. 5F)
VII 800–1,400 At this stage the nucleus (germinal vesicle) shifted toward the edge of the oocyte. Succeeding the nucleus
migration was the germinal vesicle breakdown (Fig. 5G)

protracted (Khan et al., ’90). Peak spawning season was observed stages have been classified into four stages (Ravaglia and
to be November in this study based on the results of the GSI and Maggese, 2002; Nejedli et al., 2004; Koc et al., 2008), five stages
OD. Also, the presence of many large oocytes in January (Abascal and Medina, 2005; Morris et al., 2011), six stages
suggested that H. nemurus will spawn in January. However, (Chelemal et al., 2009), seven stages (Coward and Bromage, ’98;
spawning can occur at anytime during all the months based on Cek et al., 2001) or eight stages (Utoh et al., 2003; Van Der Molen
the fact that the ovaries had mostly mature oocytes during all the and Matallanas, 2004; Solomon and Ramnarine, 2007). In
months. Khan et al. (’90) also noted a peak spawning season in this study, the oocyte developmental stages were classified into
November in H. nemurus. seven stages following the histological criteria of Coward and
GSI, which indicates growth and maturation of oocyte, was Bromage (’98).
highest in November from this study. It can be inferred that The ovarian growth phases of H. nemurus as observed in this
spawning will occur during this month. Khan et al. (’90) also study were divided into three phases, primary growth, secondary
reported a high GSI in November. Based on the fact that highest growth and maturation phases This was similar to the division
values of OD and GSI were recorded in November, it can be made by Coward and Bromage (’98) and Cek et al. (2001); and
inferred that one of the spawning period of H. nemurus is in contrary to the division made by Solomon and Ramnarine (2007),
November. which were primary and secondary growth phases.
It is pertinent to have knowledge of the ovarian develop- The histological characteristics of the oocytes in different
mental stages of fish, in understanding the reproductive development stages were also similar to those of other teleosts
performance for breeding programmes. Ovarian developmental reported in the literature (Coward and Bromage, ’98; Cek et al.,
stages of some fish species have been described by some authors. 2001; Ravaglia and Maggese, 2002; Utoh et al., 2003; Nejedli
There are differences in the division of classes based on the et al., 2004; Van Der Molen and Matallanas, 2004; Abascal and
criteria used for classification. Generally, ovarian developmental Medina, 2005; Knight et al., 2007; Koc et al., 2008; Chelemal

J. Exp. Zool.
OVARIAN DEVELOPMENT OF Hemibagrus nemurus 541

Figure 6. Electron micrograph of oocyte and ovary.

The mode of ovarian development of H. nemurus was

asynchronous, based on histological examination. Results
obtained from the scanning electron microscopy also support
the fact that H. nemurus has asynchronous mode of ovarian
development. This was evident in the electron micrograph of
mature ovary of H. nemurus. Asynchronous mode of ovarian
development was also observed in the zebrafish (Danio rerio),
European pilchard (Sardina pilchardus), blue fin tuna (Thunnus
thynnus) and lion fish (Pterois miles) (Nejedli et al., 2004; Abascal
and Medina, 2005; Knight et al., 2007; Koc et al., 2008; Morris
et al., 2011). On the contrary, synchronous mode of ovarian
Figure 5. Oocytes developmental stages of H. nemurus. development was observed in Antarctic spiny plunder fish
(Harpagifer spinosus) (Van Der Molen and Matallanas, 2004)
et al., 2009; Chellappa et al., 2010; Mohamed, 2010; Morris et al., and group synchronous ovarian development was observed in
2011). the white mullet (Mugil curema) (Solomon and Ramnarine, 2007).

J. Exp. Zool.
542 ADEBIYI ET AL.

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