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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 ( *singhij2@gmail.com)

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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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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