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American Journal of  
Life Science and Innovation (AJLSI)

Reproductive Biology and Histological Pattern of  Hilsa Shad (Tenualosa ilisha) in Bangladesh
Bimal Chandra Das1, Md. Moniruzzaman2, Mousumi Akhter3, Sabequn Nahar Swati4, Tahamina Khatun5,

Md. Mizanur Rahman6, Md. Hashibur Rahman7*, Harunur Rashid8

Volume 2 Issue 3, Year 2023
ISSN: 2833-1397 (Online)

DOI: https://doi.org/10.54536/ajlsi.v2i3.1973
https://journals.e-palli.com/home/index.php/ajlsi

Article Information ABSTRACT

Received: August 01, 2023

Accepted: September 06, 2023

Published: September 15, 2023

An experiment was carried out to identify the peak breeding season of  Hilsa in Bangladesh 
using gonadal histology through the standard procedure. A total 480 Samples were collected 
from Meghna River, Chandpur, from July 2012 to June 2013 and Tentulia river, Barisal, from 
June 2013 to June 2014. Histological sections of  gonads were analyzed and ovarian and 
testicular stages were determined according to the OECD Guidance Document for Diagnosis 
of  Histopathology of  Fish Gonads. Maturing germ cells were observed every month where 
a higher amount of  mature germ cells was found in December, January, and September to 
October, with a high in October. From February to June, the mature germ cells were less. 
Histological observations of  testis suggested that T. ilisha spawns throughout the year, but 
significant spawning takes place in September-October and December-January with a high 
abundance of  SPT (spermatids). During October, mature germ cells were mostly evident, 
and SPZ (spermatozoa) was in high proportion inside the LU (lumen of  tubules). The 
gradual maturation of  germ cells from early to ripened stages inside the testicular LU and 
peak maturity came during October. Females having ovaries at mature stages were considered 
those spawning or near to spawn. The occurrence of  mature ovaries examined by external 
characteristics began in January and continued up to December. Percentage (%) occurrence 
of  mature ovaries started to increase in February, peaked in October (75%), and decreased in 
December, January, and April. In fish ovary, the mature stages are generally observed at the 
most advanced stage of  the ovary when the fishes are ready for spawning and when mean 
GSI values are comparatively high. In this study, advanced mature oocytes were found from 
in every month during the study period. The histological study of  oocyte development and 
examination of  external characteristics of  ovaries suggested that T. ilisha spawns throughout 
the year, but significant spawning takes place in October –November and peaks in October. 

Keywords

Histology, Gonad, Tenualosa 
Ilisha, Bangladesh

INTRODUCTION
Hilsa, being anadromous, lives at sea for most of  its life 
but migrates to freshwater rivers for spawning, after 
which it returns to the sea. The Hilsa shad occurs in the 
foreshore areas, estuaries, and freshwater rivers of  the 
western division of  the Indo-Pacific region. Its marine 
distribution extends from Iran and Iraq in the Persian 
Gulf  to the west coast of  India, in the Arabian Sea, 
and in the Bay of  Bengal, the coast of  Bangladesh and 
Myanmar. Although it is distributed in large open area, 
the major percentage of  Hilsa (about 95%) is caught by 
Bangladesh, India and Myanmar (DoF, 2020).
Bangladesh’s National fish Hilsa affords the largest 
single-species fishery in Bangladesh, especially during the 
monsoon in almost all the primary river systems, estuaries, 
and the sea area of  Bangladesh. The Hilsa fishery 
supports a commercial fishery, and in the early 1970s, it 
composed more than 95% of  the total commercial catch 
in Bangladesh (Coad et al., 2003). From the 1970s, the 
Hilsa fishery began to decline gradually, with outputs 
reaching a low point of  0.19 million tons in 2001–2002 

(BOBLME, 2012). The production of  Hilsa declines 
due to a low discharge of  water from the river Ganges 
and consequently heavy siltation in most of  the rivers, 
the gradual growth of  industries, growing urbanization, 
indiscriminate use of  fertilizers, agrochemicals, pesticides, 
and the discharge of  municipal waste are continuously 
polluting the river system. To conserve the Hilsa fishery, 
proper management measures should be developed and 
implemented. One of  the most widely practiced fishery 
management is the identification of  peak spawning season 
and the banning of  fishing accordingly. The histology of  
gonads is the most common and often the most reliable 
technique to assess the reproductive strategy and tactics 
of  fish species and is therefore applied in a large number 
of  marine laboratories. In this respect, those laboratories 
aiming to provide management advice use histology for 
maturity classification to separate the sexually immature 
component from the sexually mature component, 
fecundity studies to be used in either recruitment-related 
studies or in Egg Production Methods (to estimate 
spawning stock biomass) or to monitor the prevalence 

1 Department of  Fisheries, Barishal, Bangladesh
2 Bangladesh Fisheries Research Institute, Riverine Station, Chandpur, Bangladesh
3 Bangladesh Fisheries Research Institute, Marine Fisheries & Technology Station, Cox’s Bazar, Bangladesh
4 Department of  Youth development, Ministry of  Youth and Sports, Bangladesh
5 Upazila Fisheries Office, Department of  Fisheries, Madarganj, Jamalpur, Bangladesh
6 District Commandant’s Office, Bangladesh Ansar and VDP, Jamalpur, Bangladesh 
7 Bangladesh Fisheries Research Institute, Headquarters, Mymensingh, Bangladesh
8 Department of  Fisheries Management, Bangladesh Agricultural University, Mymensingh, Bangladesh
* Corresponding author’s e-mail: hasibkhan94bfri@gmail.com



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of  any type of  reproductive disturbance or failure to 
spawn regularly. By necessity, these different overall aims 
require different methodological approaches, although in 
many cases, the histological protocol as such is reasonably 
similar. In summary, histology is considered to be an 
important tool to better understand the productivity of  
fish stocks and thereby, in the end, contributes to the 
internationally adopted principles of  the precautionary 
or ecosystem approach to fisheries. McAdam et al. (1999) 
found GSI to be reliable in determining the reproductive 
status of  fish species that spawn once annually but are 
of  limited value when fish are protracted spawners. 
Alternatively, histology is an effective technique used 
to identify the reproductive timing of  fishes even when 
species spawn multiple times or have a low reproductive 
investment (McAdam et al., 1999). Whereas histology is 
a more reliable technique than GSI, the amount of  time 
required and cost associated with histology diminish the 
practical advantage of  the technique. The present study 
aimed to identify the peak spawning season of  Hilsa by 
gonadal histology.

MATERIALS AND METHODS
Sampling of  fish
Hilsa fish sample were collected from two different 
location of  Bangladesh, upper Meghna River at Chandpur 
and Tentulia River at Barisal district of  Bangladesh. 
Sampling was conducted during July 2012 to June 2013 
in Meghna River and July 2013 to June 2014 in Tentulia 
river. Samples were collected bought once a month and 
20 mature Hilsa samples (10 female and 10 male) were 
collected at each sampling day. Hilsa were caught in the 
river during night using gill nets primarily. All specimens 
were preserved with crushed ice in cool fish boxes and 
brought to the laboratory as soon as possible. A total 
of  480 mature fishes (240 samples from each site; 240 
female and 240 male) covering various size groups were 
collected from both sampling sites.

Gonad Collection 
The body cavity of  fish was opened carefully by scissors 
and gonads were collected with forceps carefully. Other 
constitutional units such as muscles, fat tissues, digestive 
organs and blood veins etc. were taken away properly. 
Body weight (BW) and Gonad weight (GW; both left and 

right gonads) were measured to the nearest 0.001g. Total 
Length (TL) and Gonad Length (GL) were also measured 
to the nearest 0.01cm. After weighing gonads were fixed 
with 10% buffered formalin for 24-48 h and preserved 
with 10% formalin in small vials for further investigation.

Observation of  Ovarian External Features
General features and structure, as well as month-wise 
size, shape, and color of  female gonads of  T. ilisha were 
studied during sample collection and preservation. In 
Hilsa gonads, the left gonad was larger than the right 
one, both in length and width. External features of  
both ovaries were observed by the naked eye, and under 
the magnifying glass, the various maturity stages were 
classified based on external observation of  ovaries.

Histological Observation of  Hilsa Gonads
For the histological study of  Hilsa gonad, the microscopic 
slides were prepared by the following procedure followed 
by Agarwal (1996) with slight modification.
First of  all, a piece of  buffered formalin preserved 
gonad tissue was dissected randomly from the proximal 
or medial part of  one lobe for histological examination. 
A transverse segment of  about 2-3mm thick from the 
middle part of  the right ovary was taken. After taking a 
piece of  gonad tissue following steps were followed for 
histological study.

Sample Preparation (Dehydration, Clearing, 
Infiltration, Embedding, Trimming, Sectioning, 
Staining, Mounting)
The gonads samples were kept in the dehydration cassette 
and passed through a graded series of  alcohol series kept 
in glass jars (Figure 1). The dehydration schedule was as 
follows:

Sl. No. Solution Time
1 80% Ethanol 12 hours (overnight)
2 95% Ethanol 1 hour
3 95% Ethanol 1 hour
4 100% Ethanol 1 hour
5 100% Ethanol 1 hour
6 100% Ethanol 1hour

Figure 1: Dehydration using graded alcohol series



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Dehydrated gonad samples were then cleared using 
benzene twice successively for 2 hours to remove traces 
of  alcohol to have consistent paraffin blocks.
The process was as follows:

Paraffin-embedded blocks were sectioned by a microtome 
knife in a microtome machine at 5µm thickness. The 
sections were placed on previously tagged and prepared 
glass and dried on a hot plate overnight at 38°C. The 
prepared section slides were stored in the refrigerator for 
staining (Figure.4).
Staining is a process by which samples are stained 
with various dyes and staining materials so that their 
components are visible under a microscope. The gonad 
sections were stained through routine H-E staining 
protocol as mentioned below:

Sl. No. Solution Time
1 Benzine 1 hour
2 Benzine 1 hour

The samples were then infiltrated with paraffin in a 
paraffin incubator using following steps:

Sl. No. Solution Time
1 Paraffin 40 minutes
2 Paraffin 40 minutes

After infiltration, the cassettes were taken out from an 
automatic tissue processor; being opened, two samples 
were placed in the middle of  the cassette and filled with 
melted paraffin from the wax dispenser. Then, steel covers 
were placed on each cassette and allowed to cool to room 
temperature. After cooling, the cassettes were put into a deep 
freezer for a few minutes, allowing easy steel plate removal 
from the cassettes and smooth sectioning (Figure 2).

Figure 2: Embedding using malted paraffin

Trimming is a process in which the undesirable wax 
layers of  the embedded blocks are trimmed by a knife to 
obtain suitable blocks. Trimming was done by using old 
microtome blades. Trimming allowed easy sectioning. In 
this step, both side trimming and surface trimming were 
conducted (Figure 3).

Figure 3: Trimmed tissue fixed with wooden block

Sl. 
No.

Solution Time Process

1. Xylene 10 minutes Clearing (1-3)
2. Xylene 10 minutes
3. Xylene 10 minutes
4. 100% alcohol 5 minutes Dehydration 

(4-8)5. 100% alcohol 5 minutes
6. 90% alcohol 3 minutes
7. 80% alcohol 3 minutes
8. 70% alcohol 3 minutes
9. 50% Ethyl alcohol 2 minutes Staining 

(9-15)10. Distilled water 15 dips
11. Haematoxylene 

(Mayer’s) 
3 minutes

12. Wash in tap water 15 minutes
13. 50% Ethyl alcohol 10-15 dips
14. 95% Ethyl alcohol 30 seconds
15. Eosin Y 1 minutes
16. 95% Ethyl alcohol 2 minutes Rehydration 

(16-19)17. 100% Ethyl alcohol 1 minutes
18. 100% Ethyl alcohol 3 minutes
19. 100% Ethyl alcohol 1 minutes
20. Xylene 20 minutes Clearing (20-

21)21. Xylene 20 minutes
22. Drying Over night
23. Mounting Over night

Figure 4: Staining of  sectioned gonad tissues using H-E 
protocol



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D.P.X. was used for mounting as a mounting agent. A jot 
of  D.P.X. is put on each slide, followed by the attachment 
of  the coverslip (22mm×22mm). After mounting, the 
slides were put for several hours at room temperature.

Microscopic Observation of  Gonad Tissue Slides
After mounting, the slides were observed under an 
electric microscope (Olympus) connected to the 
computer with a viewer (Magnus viewer). The viewer 
was also equipped with a camera. With the help of  this 
mechanism, numerous photographs were snapped at 
different magnifications (Figure.5).

RESULTS
External Feature of  Ovary 
The maturation of  the ovary could be explained by 
examination of  the external feature of  the ovary. Because 
the shape, size, and color of  the ovary varied considerably 
according to the degree of  maturation of  the oocytes, 
the immature ovary is small reddish, and transparent 
in nature. In mature condition, the ovary’s color turns 
yellowish and becomes larger in size.

Histological study of  Ovary of  Female Hilsa of  
Meghna River, Chandpur
Ovarian development is subdivided into distinct 
developmental stages according to physiological, 
biochemical, morphological, and histological criteria. 
Maturity stages were determined following the key 
outline described by Matsuyama et al. 1982 and Shinkafi 
et al., 2011. Different steps found in the present ovarian 
histological study are presented in the figures (Figure 7 to 
Figure 10) below.
GVB (Germinal Vesicle Breakdown) was also seen 
when female Hilsa partially spent. In the present study, 
after microscopic observation, the percent occurrence 
of  different stages of  oocytes in different months was 
calculated and presented in Table 1.

Figure 5: Microscopic observation of  stained gonad sections

Table 1: Percent (%) occurrence of  the oocyte stages of  ovarian development found in the histological study of  
Hilsa ovary during the study period July 2012 to June 2013 in the Meghna river, Chandpur.
Month EPNO LPNO YV YG PM M GVB
July 6 5 8 12 11 58 Seen
August 9 10 - - 24 47 Seen
September 7 11 12 21 - 49 -
October 4 8 - 8 7 73 Seen
November 2 5 - 9 18 66 Seen
December 27 37 - - 22 14 -
January 6 7 20 22 16 29 -
February 5 9 16 19 14 37 Seen
March 8 13 13 14 19 33 -
April 12 17 16 18 13 24 -
May 7 8 11 23 12 39 -
June 11 14 10 13 11 41 -

Here, EPNO=Early perinucleolar oocyte, LPNO=Late perinucleolar oocyte, YV=Yolk vesicle, YG=Yolk granule, PM=Pre-
mature, M=Mature

From Table 1 and Figure 6, it is extracted that the highest 
(73%) and lowest (14%) percentage of  mature oocytes 
were found in the month of  October and December, 
respectively. In the month of  July, the highest percentage 
of  oocytes was in the mature stage (58%), and the lowest 
percentage of  oocyte was in early perinucleolar oocyte 
stage (5%) along with all six oocyte developmental stages. 
In the month of  August, no yolk granular stages and yolk 
vesicle were found; however, 47% of  oocyte was in the 
mature stage, whereas 9% oocyte was in early perinucleolar 

oocyte stage, besides 10% in late perinucleolar oocyte, 24% 
pre-mature stage. In September, the percentage of  mature 
oocyte increased little (49%) with 7% early perinucleolar 
oocyte, 11% late perinucleolar oocyte, 12% yolk vesicle, 
21% yolk granular stage; however no pre-mature oocyte 
was found. In October, the highest percentage (73%) 
comprised the mature stages oocyte with 7% pre-mature, 
8% yolk granule, 8% late perinucleolar oocyte and 4% 
early perinucleolar oocyte. In November, the percentage 
of  mature oocyte continued in the highest rank (66%) with 



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2% early perinucleolar oocyte stage, 5% late perinucleolar 
oocyte, 9% yolk granule, 18% pre-mature without 
having yolk vesicle stage. In December, the percentage 
of  mature oocyte declined considerably. It reached only 
14% with no yolk granule and yolk vesicle stage oocyte, 
27% early perinucleolar oocyte, 37% late perinucleolar 
oocyte (highest in number), 22% pre-mature. In January, 
the percentage of  mature stage oocytes increased little 
by 29% with 6% early perinucleolar oocyte, 7% late 
perinuclolear oocyte, 20% yolk vesicle, 22% yolk granule, 
16% pre-mature stage. In February, the mature stage 
oocyte increased again by 37% with 5% early perinucleolar 
oocyte, 9% late perinucleolar oocyte, 16% yolk vesicle 
stage, 19% yolk granule, 14% pre-mature. In March, 
the mature stage oocyte again declined and dropped 
to 33% with 8% early perinucleolar oocyte, 13% late 
perinucleolar oocyte, 13% yolk vesicle stages, 14% yolk 
granule, 19% pre-mature stage. In April, the percentage 
of  mature oocyte continued to declining trend, reached 
to 24% this month other stages of  oocyte were 12% 
early perinucleolar oocyte, 17% late perinucleoar stage, 
16% yolk vesicle, 18% yolk granule, 13% pre-mature 

stage. In May, the percentage of  mature oocyte started to 
rise again and it found 39% with 7% early perinucleolar 
oocyte, 8% late perinucleoar stage, 11% yolk vesicle, 23% 
yolk granule, 12% pre-mature stage oocyte. In June, the 
mature oocyte percentage continued to rise and reached 
to 41% having 11% in pre-mature, 13% yolk granule and 
10% yolk vesicle, 11% early perinucleolar oocyte and 14% 
late perinucleolar oocyte.
Yolk vesicle stages were found throughout the year 
except August, October and November, December. Yolk 
granule stages were absent in August and December. Early 
perinucleolar oocytes were also observed throughout 
the year except May, September, and November. Late 
perinucleolar oocytes were observed throughout the year. 
Pre-mature oocyte appeared throughout the year except 
for September and mature oocytes appeared throughout 
the year during study period.
The advanced oocyte i.e. mature oocyte was found in 
the very month of  the study period. The occurrence of  
the mature oocyte in every month of  the study period 
confirmed that T. ilisha spawns precisely throughout the 
year, but significant spawning takes place in October.

Figure 6: Month-wise occurrence (%) of  different stages of  oocyte of  ovarian development of  female Hilsa 
collected from Meghna river during July 2012 to June 2013. [Here, EPNO=Early perinucleolar oocyte, LPNO=Late 
perinucleolar oocyte, YV=Yolk vesicle, YG=Yolk granule, PM=Pre-mature, M=Mature]



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Figure 7: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled in July 
to September, 2012 [A-B: July; C-D: August; E-F: September]. EPNO, Early perinucleolar oocyte, LPNO; Late 
perinucleolar oocyte; YV, Yolk vesicle; YG, Yolk granule; PM, Pre-mature; M, Mature stage; GVB, Germinal vesicle 
breakdown.



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Figure 8: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled in 
October to December, 2012 [A-B: October; C-D: November; E-F: December]. EPNO, Early perinuclear oocyte; 
LPNO, Late perinuclear oocyte; YG, Yolk granule; PG, primary growth oocyte; PM, Pre-mature; M, Mature stage.

Figure 9: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled in January to 
March, 2013 [A-B: January; C-D: February; E-F: March]. EPNO, Early perinucleolar oocyte; LPNO, Late perinucleolar 
oocyte; YV, Yolk vesicle; YG, Yolk granule; PM, Pre-mature; M, Mature stage; GVB, Germinal vesicle breakdown.



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Figure 10: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled in April 
to June, 2013 [A-B: April; C-D: May; E-F: June]. EPNO, Early perinucleolar oocyte; LPNO, Late perinucleolar oocyte; 
YV, Yolk vesicle; YG, Yolk granule; PG, primary growth oocytes; PM, Pre-mature, M, Mature stage.

Histological Study Reproductive Cycle of  T. Ilisha 
In the present study, the following stages of  testicular 
germ cells were observed from the month-wise (July 2012 
to June 2013) samples of  T. ilisha testes. Mature germ cells 
were less evident in July. SC, ST, and SZ were observed 
in the histo-sections under the microscope. There was 
appearance of  some empty LUs in the slide (Figure 11A). 
Mature germ cells were not found in August. SC and ST 
were observed in the histo-sections under the microscope. 
The testicular LUs were full of  SZ (Figure.11B). Mature 
germ cells were more evident in September. SC, ST 
and SZ were observed in the histo-sections under the 
microscope. The testicular LUs were full of  SZ and some 

empty lumens also found (Figure11C).
Maturing germ cells were mostly evident in October. 
ST, and SZ were observed in the histo-sections under 
microscope. ST were high in number together with 
significant accumulation of  SZ. Some lumens of  
tubules (LU) were found empty (Figure. 11D). Early and 
developing stage germ cells were evident in November. 
The empty lumen was more evident. SC and ST were less 
in the histo-sections under microscope (Figure. 11E). 
Mature germ cells were more evident in December. SZ 
were observed in the histo-sections under microscope. 
SZ more in number and some empty lumens also 
found (Figure.11F). Mature germ cells were evident in 



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Figure 11: [A-F]-Haematoxylin-eosin (H-E) staining of  tissue sections of  T. ilisha testes (at 40x magnification) 
sampled in July to December, 2012 [A: July, B: August, C: September, D: October, E: November, F: December]. SG, 
spermatogonia; ST, spermatids; SC, spermamatocytes; SZ, spermamatozoa; LU, lumen of  tubules.



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Figure 12: [A-F]-Haematoxylin-eosin (H-E) staining of  tissue sections of  T. ilisha testes (at 40x magnification) 
sampled in January to June, 2013 [A: January, B: February, C: March, D: April, E: May, F: June]. SZ, spermamatozoa; 
ST, spermamatides; SC, spermatocytes and SG, spermatogonia; LU, lumen of  tubules.

January. SZ and ST were observed in the histo-sections 
under microscope. SZ more in number and some empty 
lumens also found (Figure.12A). Empty lumens were 
more evident in February. SZ, ST and SC were observed 
in the histo-sections under a microscope. SZ were less 
in number and empty lumens were more in number 
(Figure.12B). Early and developing stage germ cells were 
mostly evident in March. SG, SC and ST were observed 
in the histological-sections under microscope. SC were 
less in number (Figure.12C). Early-stage germ cells 
were more evident in April. SC and ST were observed 
in the histo-sections under microscope. SC were high in 
number (Figure.12D). SC and ST were observed in the 
histo-sections under microscope in May. Abundance of  
STs was increased although there is a significant amount of  
SC visible under microscope (Figure.12E). Maturing germ 
cells were not found in July. SC, ST, and SZ were observed 
in the histo-sections under a microscope. SPT were high 
in number together with significant accumulation of  
SZ. Some lumens of  tubules (LU) were found empty 
(Figure.12F).

Histological Study of  the Ovary of  Female Hilsa of  
Tentulia River, Barisal
The ovary of  the female Hilsa was bi-lobed elongated 
and situated in the body cavity. The shape, size, and color 
of  the ovary varied considerably according to the degree 

of  maturation of  the oocytes; the immature ovary is 
small reddish and transparent in nature which turns into 
yellowish in ripe condition. 
Ovarian development is subdivided into distinct 
developmental stages according to physiological, 
biochemical, morphological and histological criteria. 
Maturity stages were determined following the key outline 
as described by Matsuyama et al. 1982. Different stages that 
found in the present ovarian histological study are presented 
in the figures (Figure.14 to Figure.17) and described below.
Germinal Vesicle Breakdown (GVB) also occurred 
when female Hilsa partially spent. In the present study, 
after microscopic observation, the percent occurrence 
of  different stages of  oocytes in different months were 
calculated and presented in the Table 2.
From the Table 2 and Figure. 13, it is found that the 
highest (75%) and lowest (10%) percentage of  mature 
oocyte found in the month of  October and December, 
respectively. In the month of  July, 60% oocyte was in the 
mature stage which is the highest in this month having 
5% early perinucleolar oocyte, 5% late perinucleolar 
oocyte, 10% yolk vesicle stage, 20% pre-mature, and no 
yolk granule stages. In the month of  August, no yolk 
granular stage oocytes were found; however, 40% oocyte 
was in the mature stage where as 10% oocyte was in early 
perinucleolar oocyte stage, with 15% late perinucleolar 
oocyte, 15% yolk vesicle stage, and 20% pre-mature stage. 



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In September, percentage of  mature oocyte increased 
10% that the previous month and reached to 50% with 
no yolk vesicle and early perinucleolar oocyte stage, 
10% late perinucleolar oocyte, 20% yolk granular stage, 
20% pre-mature stages oocytes. The month of  October 
comprises the highest percentage (75%) of  mature stages 
oocyte among the months with 5% pre-mature, 10% 
yolk granule, 5% late perinucleolar oocyte and 5% early 
perinucleolar oocyte. In November, the percentage of  
mature oocyte was also in highest rank (65%) however 
start decline, this month’s ovary also contained 5% late 
perinucleolar oocyte, 10% yolk granule, 20% pre-mature 
oocytes and on early perinucleolar oocyte and yolk vesicle 
stage oocyte. In December, the percentage of  mature 
oocyte reached in lowest percentage among the month 
and it was only 10%, the ovary contained 30% early 
perinucleolar oocyte, 40% late perinucleolar oocyte, 20% 

pre-mature oocytes and no yolk granule and yolk vesicle 
stage oocyte. In January, the percentage of  mature stage 
oocyte started to increase reached to 35% having 5% early 
perinucleolar oocyte, 5% late perinuclolear oocyte, 20% 
yolk vesicle, 20% yolk granule, 15% pre-mature stage 
oocytes. In February, the mature stage oocyte increases 
more 10% than January reached to 45% having 10% 
early perinucleolar oocyte, 10% late perinucleolar oocyte, 
25% yolk vesicle stage, 10% pre-mature oocytes and no 
yolk granule stages were found. In March, the mature 
stage oocyte again declines and dropped to 40% having 
10% early perinucleolar oocyte, 15% late perinucleolar 
oocyte, 20% yolk vesicle stages, 15% pre-mature stage 
oocytes and no yolk granule stages were found. In April, 
the percentage of  mature oocyte continued to declining 
trend, depleted to 25%, this month other stages of  
oocyte were found as 20% in early perinucleolar oocyte, 

Table 2: Percent (%) occurrence of  the oocyte stages of  ovarian development that found in the histological study 
of  Hilsa ovary during the study period July 2013 to June 2014.
Month EPNO LPNO YV YG PM M GVB
July 5 5 10 - 20 60 Seen
August 10 15 15 - 20 40 -
September - 10 - 20 20 50 Seen
October 5 5 - 10 5 75 -
November - 5 - 10 20 65 -
December 30 40 - - 20 10 Seen
January 5 5 20 20 15 35 -
February 10 10 25 - 10 45 -
March 10 15 20 - 15 40 Seen
April 20 - - 40 25 15 -
May - - 15 30 20 35 -
June 15 15 10 10 20 30 -

Here, EPNO=Early perinucleolar oocyte, LPNO=Late perinucleolar oocyte, YV=Yolk vesicle, YG=Yolk granule, PM=Pre-
mature, M=Mature

Figure 13: Month-wise occurrence (%) of  different stages of  oocyte of  ovarian development of  female Hilsa collected 
from Tentulia river, Barisal during July 2013 to June 2014. [Here, EPNO=Early perinucleolar oocyte, LPNO=Late 
perinucleolar oocyte, YV=Yolk vesicle, YG=Yolk granule, PM=Pre-mature, M=Mature]



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40% in yolk granule, 25% pre-mature stage and no late 
perinucleolar stages and yolk vesicle stages. In May, the 
percentage of  mature oocyte started to increase again 
and it found 35% with 15% yolk vesicle stage, 30% yolk 
granule, 20% pre-mature stage and early perinucleolar 
oocytes and late perinucleolar oocytes were not found in 
this month. In June, mature oocyte percentage stayed in 
higher rate with little bit decreased than previous month 
valued 30% with 20% pre-mature, 10% yolk granule, 10% 
yolk vesicle, 15% early perinucleolar oocyte and 15% late 
perinucleolar oocyte.
Yolk vesicle stages were not found in March, September, 

October and November, December. Yolk granule stages 
were found throughout the year except February, July, 
August, and December. Early perinucleolar oocytes were 
also observed throughout the year except, May, September, 
and November. Late perinucleolar oocytes were observed 
throughout the year except April and May. Pre-mature 
and mature oocyte appeared throughout the year during 
study period. The advanced oocyte i.e., mature oocyte was 
found in every month of  the study period. The occurrence 
of  mature oocyte in every month of  the study period 
confirmed that T. ilisha spawn precisely throughout the 
year, but major spawning take place in October.

Figure 14: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled from 
Tentulia river, Barisal in July to September, 2013 [A-B: July; C-D: August; E-F: September]. EPNO, Early perinucleolar 
oocyte; LPNO, Late perinucleolar oocyte; YV, Yolk vesicle; YG, Yolk granule; PM, Pre-mature; M, Mature; GVB, 
Germinal vesicle breakdown.



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Figure 15: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled from 
Tentulia river, Barisal in October to December, 2013 [A-B: October; C-D: November; E-F: December]. EPNO, Early 
perinuclear oocyte; LPNO, Late perinuclear oocyte; YG, Yolk granule; PG, primary growth oocyte; PM, Pre-mature, 
M, Mature stage; GVB, Germinal vesicle breakdown.



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Figure 16: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled from 
Tentulia river, Barisal in January to March, 2014 [A-B: January; C-D: February; E-F: March]. EPNO, Early perinucleolar 
oocyte; LPNO, Late perinucleolar oocyte; YV, Yolk vesicle; YG, Yolk granule; PM, Pre-mature; M, Mature stage; GVB, 
Germinal vesicle breakdown.



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Figure 17: [A-F]-Haematoxylin-eosin staining of  tissue section of  T. ilisha ovary at 4x magnification sampled from 
Tentulia river, Barisal in April to June, 2014 [A-B: April; C-D: May; E-F: June]. EPNO, Early perinucleolar oocyte; 
LPNO, Late perinucleolar oocyte; YV, Yolk vesicle; YG, Yolk granule; PM, Pre-mature; M, Mature stages; PG, primary 
growth oocytes.

DISCUSSION
The reproductive activity in T. ilisha is continuous, and 
the presence of  spermatogenic and oogenic cells under 
their particular development stages was observed in all 
specimens examined monthly over two years of  study 
(Andrade et al., 2001). This pattern is also described for 
other teleosts, as a result of  tropical water conditions 
(Andrade et al., 2001; Lowe-McConnell, 1987). Study of  
vitellogenic stage (VG) of  oocyte and increased gonad 
weight (GW) and histology of  gonad are the important 
aspects of  reproductive biology that indicate the 
maturation level and can estimate approximate spawning 
time (Brown-Peterson et al., 2011). In the comparison of  
ovarian development with age from the previous study 
by Ahmed et al. (2020), Hilsa from the Meghna River, the 
Bay of  Bengal, the Padma River, and the Tetulia River 
showed regular ovarian development in respect to their 
age. No fish were found with a fully matured ovary at the 
age below 2 years.
In the teleost fishes, the process of  oogenesis may be 
divided into five to eight stages (Andrade et al., 2001). 
In the present study, stages of  gonadal development 
in Hilsa shad were identified by histological research 
according to the scales described by Shinkafi et al. (2011) 
and divided into six stages; such as early perinucleolar 
oocyte (EPNO), late perinucleolar oocyte (LPNO), yolk 
vesicle (YV), yolk granule (YG), Pre-mature (PM) and 
mature (M) stages. PG, primary growth oocytes. Besides 
these six significant stages, other two stages, such as 
germinal vesicle breakdown (GVB) and primary growth 
oocytes (PG) also found in a few cases. Panhwar et al. 
(2011) studied reproductive patterns and some biological 
features of  anadromous fish T. ilisha from Pakistan. 
They identified five stages of  oogenesis, nearly ripe, 
fully developed, running ripe, partially spent, and spent. 
Lee et al. (2005) studied with Leiognathu sequulus and they 
found eight maturity stages of  oocytes as chromatin 
nucleus, perinucleus yolk vesicle, primary yolk, secondary 
yolk, tertiary yolk, migratory nucleus and ripe stage by 

histological examination of  ovaries.
On the other hand, the four different spermatogenic 
stages were identified in the T. ilisha as described by 
Andrade et al., 2001. The stages are Spermatogonia (SG), 
Spermatocytes (SC), Spermatids (ST), Spermamatozoa 
(SZ). From the histological study, it was found that T. 
ilisha testis contains early and developing stage germ cells 
from April-June. Maturing germ cells were observed every 
month where a higher amount of  mature germ cells were 
found in December, January and September to October 
with a high in October. In the month of  February to June, 
the mature germ cells were less. Histological observations 
of  testis suggested that T. ilisha spawns throughout the 
year, but major spawning occurs in September-October 
and December-January with a high abundance of  ST. 
During October, mature germ cells were mostly evident 
and SZ was in high proportion inside the LU. The 
gradual maturation of  germ cells from early to ripened 
stages inside the   testicular LU and peak maturity during 
October is indicative of  the fact that the peak spawning 
season of  T. ilisha species is during October. The findings 
on testicular cycles coincide with that of  ovarian cycles 
of  this fish which confirms the fact that T. ilisha breed 
throughout the year and the peak breeding season in 
October. Some other studies on testicular development 
have been done on different fishes of  Bangladesh, 
they also identified four testicular developmental 
stages. Siddiqua et al. (2000) observed the testicular 
development in O. pabda and identified three stages of  
sperm formation, namely spermatocytes, spermatids, and 
spermatozoa. The percent distribution of  spermatozoa 
was highest in July (about 92%). By analyzing the 
histology of  spermatogenesis, it was established that this 
species breeds once a year. Akhter (2011) found that early 
and developing germ cells in Sperata seenghala mainly were 
observed during April- May and mature germ cells found 
in July-August. Maya (2011) studied on gametogenesis 
of  Mystus cavasius and found that mature germ cells (ST 
and SZ) were observed from July to August samples of  



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the testis. Alam (2009) observed a large amount of  ST, 
SZ and small amount of  SC in testis of  O. pabo sampled 
from April to June. Testicular LU was full of  SZ in June 
samples, indicating the peak breeding season of  O. pabo. 
In the present study, mature oocytes were observed each 
month. The percentage of  mature oocytes stated to 
increase in May and continued to November, with peaked 
in October (73% in the Meghna River and 75% in the 
Tentulia River) and decreased in December (lowest ever 
mature oocyte among the months; 13% in the Meghna 
River and 10% in the Tentulia River), January, and April. 
Histological examination and presence of  mature in 
oocyte throughout the year suggested that T. ilisha spawn 
throughout the year but major spawning takes place in 
October. On the other hand, a higher percentage of  
mature oocytes from May to November suggested the 
prolonged spawning season of  Hilsa shad. This study 
result agreed with a few previous studies. Pillay et al. 
(1963) studied with T. ilisha of  Saurashtra coast and stated 
five ovarian development stages as immature, maturing, 
mature, partly spent, and spent. Wallac and Selman (1981) 
noted that the ovary of  T. ilisha was the synchronous type 
that contained almost similar oocytes maturation stages. 
This type of  oocyte maturity was the most common 
strategy among teleosts. In this study, advanced, mature 
oocytes were found every month during the study period. 
The study indicated T. ilisha breeds throughout the year, 
with a peak in October.

CONCLUSION
Gonadal histology of  teleost fishes is the most common 
and reliable technique to assess the reproductive strategy 
of  fishes. The present study of  the histological study of  
both female and male gonads suggested that T. ilisha has 
a prolong spawning season with a peak in the month of  
October. 

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