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Ovarian development in Labeo dyocheilus (McClelland) during active
reproductive phase under captive and wild conditions
Ajeet Singh1, I.J. Singh*1, R.N. Ram1 and B. Kushwaha2
1Department of Fishery Biology, College of Fisheries, G.B. Pant University of Agriculture and Technology,
Pantnagar-263 145, India2National Bureau of Fish Genetic Resources, Canal Ring Road, Dilkusha, Lucknow-226 002, India
(Received: June 09, 2005 ; Revised received: August 17, 2006 ; Accepted: September 09, 2006)
Abstract: Ovarian development in Labeo dyocheilus was assessed during active reproductive phase under ambient environmental conditions in captivity and
wild. Increasing day length and water temperature seemed favourable for ovarian development in female L. dyocheilus under both conditions. Gonadosomatic
index (GSI) was lower in May and higher in July in captivity (6.168 and 13.366) and wild (5.798 and 16.166 ) respectively. Ovarian development started little
bit in advance in captivity with late yolk vesicle stage oocyte in May when yolk globule stage oocytes were prominent in wild. Fully grown oocytes with germinal
vesicle migration (GVM) and germinal vesicle breakdown (GVBD) stages were observed in July in both conditions. Degree of transformation of developing
oogonials into advance stage oocytes was observed to be better in wild fish compared to captivity reared ones. The histophysiological changes in liver
corresponded well to the progression of ovarian development. Successive granulation and vacuolization of hepatocyte cytoplasm were indicative of
augmented synthetic activity and probably mobilization of energy content for oocyte growth. These observations indicated that normal ovarian development
of L. dyocheilus under captivity in Tarai region of Uttarakhand would be useful for success of its seed production in captivity for stock augmentation in wild
or species diversification in aquaculture.
Key words: Ovary, Liver, Day length, Temperature, Labeo dyocheilus, Hepatosomatic Index, Gonadosomatic index
PDF of full length paper is available with author ( *[email protected])
Introduction
Success of reproduction dependents upon normal gonadal
development stimulated by favorable environmental conditions.
Photoperiod and temperature are most important factors for
controlling the reproductive activities in most of the seasonally
breeding teleosts (Lam, 1983; Shankar et al., 2007) including
cyprinids (Hontela and Stacey, 1990). Gonadosomatic index (GSI)
is generally used as a reliable criterion for expression of gonadal
development and reproductive effort in fishes (Calow, 1979;
Saksena, 1987). Hepatosomatic index (HSI) has been often used
as indicator of energy status in relation to gonadal development and
growth of fish (Wootton et al., 1978 ; Campbell and Love, 1978;
Shankar and Kulkarni, 2007).
Oogenesis is the process of transformation of primordial
germ cells (PGCs) into ova, ready to be fertilized followed by
embryonic development. Normally six oogenesis stages i.e.
formation of PGCs, transformation of PGCs into oogonia and
transformation of oogonia into oocytes (onset of meiosis), vitellogenic
growth of oocytes while under meiotic arrest, resumption of meiosis
(maturation) and expulsion of the ovum from its follicle (ovulation)
are associated with gonadal development and maturation in several
teleosts (Patino and Sullivan, 2002), whereas nine maturity stages
were described for gonadal development in Mediterranean
amberjack (Marino et al., 1995). Captive reared females failed to
not only spawn, but also to complete vitellogenesis and oocyte
maturation (Micale and Perdichizzi, 1994; Micale et al., 1996, 1999).
Failure of spawning in captivity reared fish was much related with
the arrest of oocyte development at tertiary yolk stage as compared
to wild fish (Lee and Yang, 2002).
Histological changes in liver (Olivereau and Olivereau,
1979; Medda et al., 1980; Tam et al., 1983; Singh et al., 2005) and
HSI (Singh and Singh, 1983,1984 ; Singh et al., 2004) have been
correlated with gonadal development and maturation in several
female fishes. Similar information is not available for L. dyocheilus,
hence, the present investigation was undertaken to assess its ovarian
development under captive and wild conditions.
Materials and Methods
Adult live specimens of L. dyocheilus, collected during 2002
from Domunda and Marchula (river Ramganga) and Tiger top and
Kosi barrage (river Kosi) of Uttarakhand hills, were brought to the
College of Fisheries, Pantnagar and stocked in earthen ponds
(captivity). Fish were fed @ 5% body weight twice daily (morning
and evening) with conventional carp feed prepared by mixing rice
bran and oil cake in the ratio of 1:1. Fish from wild were collected
during May to August 2003 for study of gonadal development. For
histological studies and assessing GSI and HSI levels in relation to
ovarian development after recording body weight, ovary and liver
samples from female specimens of L. dyocheilus were collected
each month during May to August, 2003 from wild or reared in
captivity. GSI and HSI were calculated as percentage of weight of
ovary and liver in relation to body weight. Small portions of ovary
Journal of Environmental Biology March 2008, 29(2) 169-174 (2008)
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170
and liver were fixed in Bouin’s solution for histological studies. Paraffin
sections were cut at 5µm thickness and stained with hematoxylin-eosin
for histological observations prior to microphotography (Humason,
1979). Monthly data of atmospheric temperature, day length and
rain fall were obtained from Agro-Meteorology department of the
University while pond water temperature, pH and dissolved oxygen
were regularly recorded. Monthly observations of GSI and HSI
were tested for statistical significance by Two-way ANOVA using
complete randomized design (CRD) and randomized block design
(RBD) developed by Chandel (2002).
Results and Discussion
During study period, no abrupt changes in climatic conditions
were observed except total rainfall. Ambient temperature ranged
between 22.2 and 38.0oC. Total rainfall was recorded maximum in
the month of August (573 mm) while it was minimum in May (46. 2
mm). Average water temperature ranged between 21.1 and 27.9oC
and no marked change was recorded in pH and dissolved oxygen
of pond water. Meteorological data and water quality parameters
like temperature, pH and dissolved oxygen in pond water are
summarized in Table 1.
The gonadal development was almost similar in female of
L. dyocheilus reared in captivity or collected from wild during May
to August as reflected by changes in GSI values (Table 2). GSI
increased gradually from May to July and decreased in August in
females of both groups. GSI was higher in captive reared female in
May but from June to August it was significantly higher (p<0.01) in
wild collected females compared to captive reared ones. GSI ranged
from 6.168 to 13.366 in captive reared and 5.798 to 16.166 in wild
groups. Monthly variations in GSI were also significant (p<0.01) in
both the groups but these changes exhibited insignificant correlation
with rearing conditions (Table 3).
The HSI showed negative relationship with GSI in female of
both groups (Table 2). HSI was lowest in May (0.620) in captive
reared fish and highest in June (0.760) and in wild collected females it
was lowest (0.576) in July and highest (0.868) in August. A gradual
decrease in HSI was observed from May to July in wild collected
females which increased sharply in August. HSI differed significantly
(p<0.01) between wild and captive reared females and also varied
significantly (p<0.01) in relation to months in both the groups. It was
higher in June and July in captive stock and in May and August in wild
ones. The difference in HSI of both groups showed significantly (p<0.05)
correlation between captive and natural habitat conditions (Table 3).
In the month of May, ovary in captive reared female was
dominated by late yolk vesicle stage oocyte along with perinucleolar
Table - 2: Correlative changes in GSI and HSI in adult female of L. dyocheilus reared in captivity and collected from wild
Group Parameters May June July August
Captive GSI 6.168 ±0.420 11.192 ±1.892 13.366 ±0.919 10.156 ±1.166
HSI 0.620 ±0.054 0.760 ±0.101 0.675 ±0.094 0.733 ±0.016
Wild GSI 5.798 ±0.264 12.102 ±2.341 16.166 ±1.821 13.573 ±1.432
HSI 0.696 ±0.006 0.638 ±0.061 0.576 ±0.010 0.868 ±0.043
Values are mean ± SD
Table - 1: Meteorological parameters and physico-chemical characteristics of pond water during May to August, 2003
Meteorological parameters at Pantnagar Physico-chemical characteristics of pond water
MonthRange and average Range and average Total rainfall Day length Temperature pH DO (ppm)
of max. temp. (0C) of min. temp. (0C) (mm) (hr) (oC)
May 36.0-41.1 (38.0) 18.9-22.7 (22.2) 46.2 9.7 25.1 7.7 7.6
June 32.3-37.3 (34.3) 24.5-26.8 (25.3) 313.0 4.9 27.2 7.3 8.4
July 29.3-37.5 (33.0) 23.9-26.3 (25.5) 538.4 6.3 27.9 7.9 8.0
Aug. 30.9-33.1 (31.8) 24.9-25.9 (25.2) 573.0 5.2 26.8 7.2 6.8
Source: Department of Agro-Meteorology, College of Agriculture, GBPUA and T, Pantnagar
Table - 3: ANOVA-matrix for GSI and HSI of captivity reared and wild collected specimens of L. dyocheilus
Source / Index GSI HSI
DF SS MS F-value DF SS MS F-value
Treatment (a) 1.0 17.115 17.115 8.105** 1.0 0.460 E-04 0.460 0.680**
Factors (b) 3.0 243.016 81.005 38.361** 3.0 0.104 0.348 5.147**
(a*b) 3.0 13.598 4.532 2.146ns 3.0 0.729 E-01 0.243 3.591*
a = Captive and wild conditions, b = Different months, a*b = Correlation between a and b
ns = Non-significant, * = Significant at 5%, ** = Significant at 1%
Singh et al.
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171Ovarian development in Labeo dyocheilus
Fig. 1: Ovary of the captive reared fish showing dominance of yolk vesicle stage oocytes (→) and presence of oogonials (>),Immature oocytes (�) in
the month of May. HE X 45
Fig. 2: Ovary of fish collected from natural habitat showing dominance of yolk globule stage oocytes (→) and occasional presence of immature oogonials
(>) in month of May. HE X 45
Fig. 3: Ovary of the captive reared fish showing marked presence of late yolk vesicle stage oocyte (→) and fully grown oocytes (⇒) in month of June.
HE X 45
Fig. 4: Ovary of fish collected from natural habitat showing dominance of yolk globule (→) and fully grown (⇒) stage oocytes and occasionally oocytes
with germinal vesicle movement (>) in month of June. HE X 45
Fig. 5: Ovary of the captive reared fish showing dominance of fully grown oocytes (⇒) with well differentiated follicular layer (>) in month of July. HE X 45
Fig. 6: Ovary of fish collected from natural habitat showing dominance of mature stage oocytes (⇒) with separation of follicular wall (>) in month of July.
HE X 45
and early yolk vesicle stage oocytes. Oogonials and immature
oocytes were also present in moderate number (Fig. 1) while the
ovary of female collected from wild possessed significant number of
yolk globule stage oocytes along with a few oogonials (Fig. 2). In
June ovaries were full of late yolk vesicle and yolk globule stage
oocytes with randomly distributed cortical alveoli in cytoplasm (Fig. 3)
in female reared in captivity while yolk globule stage oocytes with
GVM and GVBD were observed in female of wild group ovary
(Fig. 4). Peak ovarian development with dominance of fully grown/
maturation stage oocytes with well developed follicular layer were
recorded in ovary of female of captive group in July (Fig. 5). Oocytes
exhibited similar developmental events in wild group also in July
(Fig. 6). In August, ovaries had persistent dominance of fully grown
oocytes packed with yolk mass and disappearance of cortical alveoli
in females of captive group (Fig. 7) while in wild group fully grown
oocytes advanced towards ovulatory stage (Fig. 8).
Hepatocytes in liver exhibited active biosynthetic stage
packed with cytoplasmic content in both captive reared and wild
collected females in May (Fig. 9, 10). In June, hepatocytes in captive
group showed cytoplasmic vacuolization attributable to mobilization
of cytoplasmic content (Fig. 11) but in wild group mobilization of
energy reserve seemed slightly delayed (Fig. 12).In July liver cells
of females of both, captive and wild groups were in exhaustive
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Fig. 7: Ovary of the captive reared specimen containing fully grown oocytes (⇒) and presence of immature ( ) and early vitellogenic stage oocytes (>)
in month of August. HE X 45
Fig. 8: Ovary of fish collected from natural habitat showing dominance of mature stage oocytes (⇒) with follicular wall separation and germinal vesicle
migration (>) in August. HE X 45
Fig. 9 : Active hepatocytes with rounded large nuclei and nucleoli (>) of liver of captive reared specimens in month of May. HE X 450
Fig. 10 : Large active hepatocytes of liver of specimen collected from natural habitat showing cytoplasmic granulation (>). HE X 450
Fig. 11 : Hepatocytes of liver of captive reared specimen exhibiting vacuolization of cytoplasm (>) in month of July. HE X 450
Fig. 12 : Hepatocytes of liver of specimen collected from natural habitat containing darkly stained cytoplasmic material (>) in month of June. HE X 450
condition as indicated by degranulation and decreased nucleolar
size (Fig. 13, 14). However, reorganization of liver cells with darkly
stained residual synthetic material was evident in females reared in
captivity indicating initiation of growth phase (Fig. 15) but exhaustive
condition still persisted in liver cells of female collected from wild
(Fig. 16).
In most of monsoon breeding teleosts especially cyprinids
progressive stages of gonadal development in Indian subtropical
regions are well correlated with environmental cues particularly
increasing daylength and temperature. In female L. dyocheilus from
either captive or wild conditions, increasing level of gonadal
development appeared positively correlated with increasing
daylength and temperature till ovarian maturity but lowering of
temperature associated with rainfall seemed to be crucial factor for
final oocyte maturation and subsequent processes. A positive
relationship between increasing temperature and daylength with
gonadal development during preparatory and prespawning phases
and fall of temperature due to rainfall associated with upsurge in
gonadotropin level during spawning phase has been reported for
the Indian major carp, Cirrhinus mrigala (Singh and Singh, 1984).
Like wise gonadal development and maturation in an other major
carp, Labeo rohita was found to be influenced positively by increasing
day length and temperature (Kumar et al., 2003; Singh et al., 2005).
The gonadosomatic index (GSI) is correlated positively
with gonadal development and more often is used as reliable indicator
of its maturity (Rae and Calvo, 1995). Hepatosomatic index, used
Singh et al.
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173Ovarian development in Labeo dyocheilus
Fig. 13 : Hepatocytes of captive reared specimen showing degranulation and decreased nuclear size (>) in month of July. HE X 450
Fig. 14 : Hepatocytes of liver of specimens collected from natural habitat showing intense cytoplasmic vacuolization and presence of smaller nucleoli (>)
in month of July. HE X 450
Fig. 15 : Hepatocytes of captive reared specimen containing prominent nuclei showing residual synthetic activity (>) and also small inactive hepatocytes
(>) in month of August. HE X 450
Fig. 16 : Hepatocytes of liver of specimens collected from natural habitat showing scanty cytoplasmic and nuclear content (>) in month of August. HE X
450
as indicator of energy status (Wootton et al., 1978), has been
observed to exhibit negative correlation with GSI (Singh and Singh,
1983, 1984; Kumar et al., 2001; Singh et al., 2005) suggesting
mobilization of energy stored from liver to ovary for egg production
(Rae and Calvo, 1995). In L. dyocheilus female also, like other
cyprinids of the region, HSI exhibited negative correlation with
changes in GSI in specimens of both groups despite having variation
in monthwise level of changes between the groups.
Transformation of immature and less developed oocytes into
developed and mature stage oocytes is associated with the
accumulation of lipid globules in preparatory and prespawning
seasons. Failure of completion of vitellogenesis and oocyte maturation
(Micale and Perdichizzi, 1994; Micale et al., 1996, 1999) and arrest
of oocyte development in tertiary yolk stage (Lee and Yang, 2002)
were reported in some captive reared female fishes. Despite slight
variation with regard to timing the presence of developmental and
maturational oocyte stages particularly yolk vesicle and globule stages,
yolk globule stage oocytes with GVM and GVBD in both captive and
wild group of specimens of L. dyocheilus clearly demonstrated that
ovarian development in this fish was normal under captive conditions.
Similar sequences of oocyte development processes were reported
in the Indian major carp, L. rohita in captivity under similar ambient
environmental conditions (Singh et al., 2005). Conversion of oocyte
into ova exhibited nine stages starting from basic oogonial cell type to
final mature ovum with post ovulatory follicles in Mediterranean
amberjack, Seriola dumerilli (Marino et al., 1995). Oocytes with
peripheral cortical alveoli, lobated nucleus and several nucleoli
transformed into large hydrated oocytes with increased number and
size of yolk granules during pre-spawning and spawning period in
common dentex, Dentex dentex (Loir et al., 2001).
Increased cell and nuclear size, cytoplasmic granulation
and vacuolization in hepatocytes during pre-spawning and
spawning phases in L. dyocheilus exhibited biosynthetic activity
and apparent correlation with ovarian development. In oestrogen
treated freshwater eel, Anguilla anguilla (Olivereau and Olivereau,
1979) and red grouper, Epinephelus akara (Tam et al., 1983) an
increase in the hepatosomatic index, enlargement of hepatocytes
and their nuclear size and vacuolization in cytoplasm were correlated
with biosynthetic activity.
Correlative changes in GSI and HSI, histological features
of ovary and liver in captive reared and wild group of fish, L. dyocheilus,
demonstrated that ovarian development and maturation was normal
in captivity in this fish. Normal ovarian development and maturation in
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174
female L. dyocheilus, an important food fish, under captive condition
in tarai region of Uttarakhand has wide scope for application in
itscontrolled quality seed production for stock supplementation innature/ wild and species diversification under culture system.
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