EFFECT OF THE NaCl AND NaOH ON SPERM

QUALITY AND MOTILITY OF THAI PANGAS,

Pangasius sutchi IN BANGLADESH

MS Thesis

DEPARTMENT OF FISHERIES BIOLOGY AND GENETICS

PATUAKHALI SCIENCE AND TECHNOLOGY UNIVERSITY
DUMKI, PATUAKHALI, BANGLADESH

May 2021
EFFECT OF THE NaCl AND NaOH ON SPERM
QUALITY AND MOTILITY OF THAI PANGAS,
Pangasius sutchi IN BANGLADESH

A Thesis

Submitted to
Patuakhali Science and Technology University, Patuakhali
In Partial Fulfillment of the Requirements
for the Degree of
Masters of science (MS)
in
Fisheries Biology and Genetics

By
Id No: FBG JJ 20/003
Registration No: 05763 Session: 2020-21

May 2021

i
 EFFECT OF THE NaCl AND NaOH ON SPERM
QUALITY AND MOTILITY OF THAI PANGAS,
Pangasius sutchi IN BANGLADESH

A Thesis

Submitted to
Patuakhali Science and Technology University, Patuakhali
In Partial Fulfillment of the Requirements for the Degree of
Masters of Science (MS)
in
Fisheries Biology and Genetics

Approved as to the style and contents by

……………………………………………………………………….. ………………………………………………………………………..

Professor Dr. Md. Ariful Alam Professor Dr. Muhammad A.B. Siddik
Supervisor Co-Supervisor

…………………………………………………………………………………..

Professor Dr. Muhammad A.B. Siddik

Chairman,
Department of Fisheries Biology and Genetics
Patuakhali Science and Technology University, Patuakhali

May 2021
ii
 DECLARATION

I declare that the thesis hereby submitted by me for MS degree at the Patuakhali Science and
Technology University is my own independent work and has not previously been submitted
by me at another university/faculty for any degree.

Date: …………………… The Author

iii
 Dedicated
to
My Beloved Parents
And
Respected Teachers
 Acknowledgements

The author expresses his gratitude and praises to the almighty Allah who enabled him to
pursue studies as well as to submit the report for the degree of Masters of Science (M.S.) in
Aquaculture.
The author feels fortunate and great pleasure to get Professor Dr. Md. Ariful Alam,
Department of Fisheries Biology and Genetics, Faculty of Fisheries, Patuakhali Science and
Technology University, Dumki, Patuakhali-8602, for, his scholastic supervision, pretty
guidance, affectionate support, and immense help throughout the entire period of planning,
conducting, and completing research work and preparation of the report.

The author feels great pleasure in expressing his deep sense of respect and gratitude, sincere
appreciation, and indebtedness to his Co-supervisor Professor Dr. Muhammad A.B. Siddik,
Chairman, Department of Fisheries Biology and Genetics, Patuakhali Science and
Technology University, Dumki, Patuakhali-8602 for his constructive suggestion and
encouragement throughout the whole period of this piece of research.
The author finds an opportunity to express his sincere appreciation and immense
indebtedness to Md. Rajib Sarkar, Associate professor and Md. Arifur Rahman (Arifin),
Assistant professor, Department of Fisheries Biology and Genetics, Faculty of Fisheries,
Patuakhali Science and Technology University, Dumki, Patuakhali-8602, for their
illuminating suggestion for completing the research work and preparation of the report.
Finally, the author expresses his deepest respect and love to his beloved parents Sunil
Chandra Mistry and Prova Rani Mazumder for their inspirations, moral support, and
blessings, for all responsibilities, and endless love not only during the study period but also
for the entire period of life.

The Author
May 2021

v
Effect of the NaCl and NaOH on Sperm Quality and Sperm Motility
of Thai Pangas, Pangasius sutchi in Bangladesh

Abstract
The study was conducted to evaluate the effectiveness of NaCl and NaOH on sperm quality
and sperm motility of Thai Pangas, Pangasius sutchi. This study was carried out in Chanchal
Matshya Hatchery at Bauphal in Patuakhali where all necessary facilities were available. The
result showed significant differences in effectiveness of the two treatments in evaluating the
sperm quality and sperm motility. The effects of NaCl solution such as the highest number
of sperm counted as 24344 ± 1734 in T1R3 and lowest number of sperm counted as 17234 ±
1013 in T1R1. The effects of NaOH solution such as the highest number of sperm counted as
10373 ± 376 in T2R3 and lowest number of sperm counted as 7219 ± 879 in T2R1. In the
case of sperm motility at 5% NaCl and 5% NaOH concentration in different time intervals
viz. 0 min, 15 min, 30 min, 60 min and 90 min after ejaculation, a significant (p < 0.05)
decreasing tendency was observed with increasing time intervals in all treatments. This study
suggested that the sperm quality and sperm motility at NaCl solution was suitable than
followed by normal running water and NaOH solution whereas 5% NaCl solution was given
the highest quality and motility of sperm of Thai Pangas. Moreover, the highest average
ovulation rate (91 ± 2.2%), fertilization rate (70 ± 0.8 %), and hatching rate (96 ± 0.8%) was
recorded at 5% NaCl concentration compared to control (normal tape water). The highest
(72%) and lowest (66%) survival of hatchlings was found at 24 h and 72 h, respectively in
normal tape water whereas highest (74%) and lowest (68%) survival of hatchlings was found
at 24 h and 72 h, respectively in 5% NaCl solution. Overall, the 5% NaCl solution was suitable
than normal running water on the basis of ovulation rate, fertilization rate of eggs, hatching
rate and survival rate of hatchling of Thai pangas. The findings of this study were provided
the basis for the potential to be a guide for further researches to evaluate the good quality
sperm of Thai pangas, Pangasius sutchi which is also helpful to apply on related species
perhaps other fishes of Bangladesh. Moreover, it was also provided the detailed information
to the hatchery managers on sperm quality and sperm motility of Thai Pangas which is helpful
to conduct successful breeding on related species perhaps other fishes under different area of
Bangladesh.

Keywords: NaCl, NaOH, Pangasius sutchi, sperm quality, sperm motility.

vi
 LIST OF CONTENTS

PAGE
CHAPTER TITLE
NO.
ABSTRACT vi
LIST OF CONTENTS vii-viii
LIST OF FIGURES ix
LIST OF TABLES x
Chapter 1 INTRODUCTION 01-09
1.1 General Introduction 02-03
1.2 Taxonomy of Thai pangas 03
1.3 Morphological Characteristics 03-04
1.4 Distribution and Habitat 04
1.5 General Information of Thai pangas 04-08
1.6 Specific objectives of the study 09
Chapter 2 REVIEW OF LITERATURE 10-14
Chapter 3 MATERIALS AND METHODS 15-20
3.1 Study Site 16
3.2 Sperm Collection 16
3.3 Experimental Design 16
3.4 Sperm Quality Assessment 17
3.5 Motility Assessment 17-18
3.6 Assessment of Life Span of Sperm 18
3.7 Analysis of Ovulation Rate (%), 19-20
Fertilization Rate (%) of Eggs, Hatching
Rate (%) and Survival Rate (%)
3.8 Statistical analysis 20
Chapter 4 RESULTS 21-25
4.1 Effects of NaCl and NaOH on Sperm 22
Quality Effects of NaCl on sperm quality
4.2 Effects of NaCl and NaOH on Sperm 22
Motility
4.3 Ovulation Rate (%) of Pangasius sutchi 22
and Fertilization Rate (%) of their Eggs
4.4 Hatching Rate (%) and Survival Rate 23-24
(%) of P. sutchi Larvae
vii
 LIST OF CONTENTS (Cont’d)

PAGE
CHAPTER TITLE
NO.
Chapter 5 DISCUSSION 26-31
Chapter 6 SUMMARY AND CONCLUSION 32-34
Chapter 7 REFERENCES 35-42

viii
 LIST OF FIGURES

FIGURE PAGE
TITLE
NO. NO.
3.1 Flow diagram of PG hormone administrated on
both male and female Thai pangas, Pangasius 18
sutchi
3.2 Flow diagram of sperm quality assessment under 19
biological microscope
4.1 Effects of NaCl on sperm motility in different 25
time interval

ix
 LIST OF TABLES

TABLE TITLE PAGE

NO. NO.
3.1 Effectiveness of NaCl and NaOH on sperm 17
quality in different % of these solutions
3.2 Single dose of PG in each male and double 17
doses of PG in each female Thai pangas,
Pangasius sutchi
4.1 Evaluation of the effectiveness of NaCl and
NaOH on sperm quality at different 23
concentration.
4.2 Evaluation of the effectiveness of NaCl and 23
NaOH on sperm motility in different time
interval
4.3 Evaluation of the average ovulation rate (%), 24
fertilization rate (%), and hatching rate (%) of
Pangasius sutchi broods, fertilized eggs, and
their hatched larvae, respectively after PG dose
administered.
4.4 Effect of PG hormone on the survival rate (%) 24
of Thai pangas, Pangasius sutchi in different
time intervals 24 h, 48 h and 72 h

x
CHAPTER 1

1
 INTRODUCTION
1.1 General Introduction
Bangladesh is considered one of the most suitable regions for fisheries in the world, with the
world’s largest flooded wetland and the third largest biodiversity in Asia after China and
India. Bangladesh, with its rich inland waters and river systems, has significant capture
fishery and aquaculture potential. Fisheries sector represents one of the most productive and
dynamic sectors in Bangladesh. The fisheries sector of Bangladesh is playing an increasingly
significant role in the economy for the last few decades. Bangladesh has achieved remarkable
progress in the fisheries sector since its independence in 1971. Fisheries sector is contributing
a very significant role in the socioeconomic development, and deserve potential for future
development in the agrarian economy of Bangladesh. According to FAO statistics 2020,
Bangladesh ranks 3rd in inland open water capture production, 5th in aquaculture production.
Bangladesh also ranks 1st in global catch of hilsa shad, illish and 4th in Tilapia in world and
3rd in Asia.

This sector is contributing significantly in food security through providing safe and quality
animal protein. The fisheries sector contributes 3.52% to GDP and 26.37% to agricultural
GDP. Fish supplements to about 60% of our daily animal protein intake (DoF, 2020). More
than 12% of populations including women are directly or indirectly engaged in various
activities under fisheries sector for their livelihood. This sector also has high potential for the
perspective of economic development of the country. Bangladesh is also blessed with vast
inland waters in the form of ponds, canals, ditches, floodplains, haors (natural depression),
baors (ox-bow lake), rivers, estuaries etc. covering an area of 47.02 lakh ha. in which ponds
cover an area of 4.04 lakh ha. Fish production from these inland water bodies during the year
2019-20 is 3832267 MT (open water capture fisheries are 1248401 MT and close water
culture fisheries are 2583866 MT) (DoF, 2020). Bangladesh earns a considerable number of
foreign currencies by exporting fish, shrimps and other fishery products that contributes
1.39% of the total national export earnings, in 2019-20, the country earns BDT 398515 lakh
by exporting almost 70.95 thousand MT of fish and fishery products (DoF, 2020).

Aquaculture has been the most rapidly growing agro-food sector in Bangladesh over the last
three decades. The overall growth performance from inland aquaculture shows a moderate,
reasonable and admirable increasing trend. The production of farmed finfish and shellfish has
been growing at a rate of 9.34% per annum over this period. The aquaculture production

2
became more than doubled (10.06 lakh MT in 2007-08 to 25.84 lakh MT in 2019-20) during
the last twelve years (DoF, 2020). In Bangladesh, aquaculture production systems are mainly
extensive and improved extensive with some semi-intensive and in very few cases intensive
systems. The present unit area aquaculture productions (MT/ha) are 5.06, 1.49, 1.93 and 1.05
for pond, seasonal waterbody, baor (oxbow lake) and shrimp gher, respectively. There are
two types of aquaculture practices are going on in Bangladesh - freshwater and coastal
aquaculture. Freshwater aquaculture comprises mainly pond farming of carps (indigenous
and exotic), pangas, tilapia, climbing perch and a number of other domesticated fish. On the
contrary, coastal aquaculture is comprised mainly of shrimp and prawn farming in ghers
(coastal pond or enclosures). In Bangladesh, on the basis of fish production of pond
aquaculture, the top 7 fish species are Thai pangas, Rui, Tilapia, Silver carp, Mrigal, Catla
and Koi (DoF, 2020). Thai pangas is one of the most important culturable fish species in
Bangladesh which contributes a significant rule in total aquaculture production.

1.2 Taxonomy of Thai pangas

Kingdom: Animalia

Phylum: Chordata

Class: Actinopterygii

Order: Siluriformes

Family: Pangasiidae

Genus: Pangasius

Species: P. sutchi

English Name: Shark catfish

Local Name: Thai pangas

1.3 Morphological Characteristics

Body is elongated and laterally compressed, without any scale. Head and abdomen are flat;
tail is constricted behind the adipose fin but a bit extended before the caudal peduncle. Head
is slightly granulated above; occipital process is used to reach to basal bone of dorsal fin;
snout is fairly prominent. Eyes are in the anterior half of the head, partly on the lower surface
of head. Mouth is sub-terminal; upper jaw is longer than the lower jaw; mouth gape is of

3
moderate size. Cleft of mouth is used to reach opposite the centre of front edge of the eye.
Four groups of teeth are present on the palate; palatine teeth are in a crescent row, vomarine
patches are separate from or nearly confluent with those on palate. Barbels are two pairs; the
maxillary pair reaches the base of pectoral fin and the mandibular pair is half as long as the
head in length. First dorsal fin is with a moderately strong spine which is strongly serrated on
its inner edge but finely serrated on its outer edge. Adipose dorsal fin is short, posteriorly
free, and originates almost opposite to the middle of the anal fin. Pectoral fin spine is serrated,
strong and as long as dorsal spine. Anal fin is large and well developed. Caudal fin is deeply
forked; upper lobe is slightly the longer. Body color is silvery, darkest along the back and
glossed with purple on sides; cheeks and the under surface of head is golden; caudal fin is
bright yellow (Talwar et al., 1991 and Day, 1878).

1.4 Distribution and Habitat

Pangasius sutchi is widely distributed in India, Bangladesh, Pakistan, Myanmar, Malaya-

peninsula, Indonesia, Vietnam, Java and Thailand (Talwar et al., 1991 and Day, 1878).
Pangasius pangasius mainly inhabits large rivers and estuaries; but can also be seen in
irrigation canals, haors, baors, beels, natural depressions and even ponds especially during
the monsoon period (Talwar et al., 1991 and Rahman, 2005).

1.5 General Information of Thai pangas

Cultured fish species refers to those species of fishes which are already introduced into the
existing fish farming system in Bangladesh. A quite large number of species are now being
used as culture species consisting both native and exotic fish species. Indigenous or native
species includes different species of major and minor carps and exotic species includes
different Chinese carps, catfishes, perches etc. Among the various culture species of catfish,
Pangasius sutchi is particularly important. Pangasius sutchi was introduced in Bangladesh
from Thailand in 1989. Later, induced breeding of this species was first accomplished
successfully in 1991 at BAU and BFRI. Because of its introduction from Thailand the fish is
popularly known as Thai pangas (Roberts and Vidthayanon, 1991). Amongst exotic fish
species, Thai pangus (Pangasius sutchi) is the best due to its easy culture system, favorable
weather condition for culture and high market demand (Sarker, 2000). The pond culture of
native pangas (Pangasius pangasius) was started in 1945 at Khulna region of Bangladesh.
But due to the lack of specific technical know-how as well as proper culture management it
was neither successful nor so popular to the local people (Sarder et al., 1994). Although

4
necessary steps for native pangas culture in closed water condition were taken in 1987 at
Chandpur, but the initiative did not face any notable success (Sarker, 2000). After the failure
of native pangas culture, the government of Bangladesh imported 100 numbers fry (0.18 g
weighted) of Thai pangus (Pangasius sutchi) from Thailand in 1990 (Sarker, 2000). Outside
Bangladesh, three important species viz. P. hypophthalmus, P. larnaudi and P. sanirwangsei
are cultured extensively in Thailand, Cambodia and Vietnam both in earthen ponds (Bardach
et al., 1972) and in floating net cages (Aguru, 1970) where it reaches up to 3 kg in two years.
The survival rate of Pangasius sutchi is satisfactory, 85%, stated by Rahman et al., 1992.
High density and semi-intensive culture of Pangasius sutchi in ponds have been established
and are very popular in Bangladesh. Such culture can produce at a rate of as high as 25-30
tons/ha/yr with protein rich diets (BFRI and BARC, 2001). Two types of culture systems have
been practiced in Bangladesh for Pangasius sutchi farming: monoculture (following intensive
culture strategy) and polyculture (following semi-intensive culture strategy). The polyculture
of carps account about 80% of the total freshwater aquaculture production in extensive and
semi-intensive system of Bangladesh (ADB, 2005 and Ahmed, 2005). The remaining 20%
are mainly from pangasius, tilapia, small indigenous species (SIS) of fish and rice-fish
farming (Muir, 2003).

However, the culture system is spreading to an increasing number of districts and has rapidly
evolved into an economically significant activity with long backward and forward linkages
providing diverse livelihood opportunities for a wide range of value chain actors (Haque,
2009). This rapid growth has occurred in part because Thai pangas is popular among fish
farmers due to the ease with which it can be cultured; possessing hardy characteristics, good
survival rates, fast growth and ability to survive at high stocking densities (Sarker, 2000). The
fish has also proven popular among consumers due to its low market value (a function of high
yields), low per unit production costs and supply, making it one of the most important cultured
species, particularly among the poor in urban areas (Belton et al. 2011b). Then the artificial
propagation practiced over the country and it gained much popularity in Bangladesh because
of its rapid growth, ease culture technique, high disease resistant and tolerance to a wide range
of environmental adaptation (Bardach et al., 1972). Pangasius sutchi is well accepted by a
wide range of people and therefore, it has been a good source of protein and calorie for poor,
medium and better-off people in rural as well as urban areas. People of this country were
economically benefited from this industry. It has gained a momentum in Bogra, Jessore,
Noakhali, and Dhaka and mainly in Mymensingh district for high growth, high demand and

5
high market price. Farming has started commercially by a private enterprise named Al-Falah
Fisheries since 1993 in Bailor Union under Trishal Upazila of Mymensingh district.
Afterwards, it was steadily expanding from Trishal to the neighboring villages and other
districts. Later, particularly in the last decades, Pangas culture was spreading to an increasing
number of districts and has rapidly evolved into an economically significant activity with
long backward and forward linkages providing diverse livelihood opportunities for a wide
range of value chain actors (Haque, 2009). Pangasius catfish is one of world’s fastest growing
fresh water species in aquaculture (Jeyakumari et al., 2016). Pangasius is now traded to well
over 100 countries worldwide as skinless and bone less fillets popularly along with portions,
steaks, fillets and its added value products (Thi et al., 2013). The industry has expanded in
terms of both production and trade. This rapid growth has occurred due to its popularity to
the pond farmers for possessing hardy characteristics, higher survival rates, fast growth, and
ability to survive at high stocking densities. The species has also proven popular among
consumers due to its low market value, making it one of the most important cultured species,
particularly among the poor in urban areas. It is therefore an important issue of addressing
Thai pangas as single species aquaculture being recognized as “Pangasius aquaculture” in
Bangladesh.

Thai pangas has grown in importance for Bangladesh's national food security as a result of its
high production rates and affordability for consumers in lower socioeconomic brackets.
Market prices are low when compared to those of Indian big carp, which still make up the
majority of Bangladesh's aquaculture output and sell for around twice as much at retail
(Belton et al. 2011b). Despite an overall rising trend, the business is subject to seasonal and
cyclical excess and underproduction. For instance, a pangasius glut in 2008 temporarily drove
down farmgate prices to levels below production costs (Edwards & Hossain 2010), but the
industry recovered in 2009. Although some worries have been voiced about the social and
environmental sustainability of pangasius aquaculture in Vietnam, these claims have largely
been disproven in that country's context. They also don't apply to catfish farming in
Bangladesh, where the marketing of fingerling and food fish, the production and distribution
of feed, and other related services significantly increase employment opportunities for people
living in rural and peri-urban areas while having a relatively small negative impact on the
environment (Belton et al. 2011a). Based on the findings of research conducted in
Bangladesh's Mymensingh region in 2009, the remainder of this article develops these topics

6
to provide a current overview of the characteristics, status, and consequences of pangasius
aquaculture.

Thai pangas aquaculture has a favorable influence on both farmers and non-farmers living in
and around the producing villages in terms of the availability of protein-rich food (Ali and
Haque, 2011). It was discovered to be a less expensive source of protein than other fish, meat,
and dal for non-farmers who are impoverished (lentil soup). This shows that low-income
individuals have simple access to locally produced, high-protein foods. Due to its better
survival, higher productivity, regular/partial harvesting, and sale, Thai pangas aquaculture
tends to make the fish available to the community members for extended periods of time
throughout the year. As a result, poor people can purchase fish with their meager cash,
whereas they cannot purchase carps or beef. Thai pangas is a resilient fish that is typically
marketed and delivered to markets alive. Since this fish can be purchased live, it received a
better score for freshness than other perishable foods. The taste of Thai pangas was rated
more favorably by the non-farmer group of persons than by the group of farmers.
Additionally, both groups gave the Pangas nearly similar marks in terms of its liking for the
kids. Due to its superior taste and lack of intramuscular bones, children like this fish (Anwar,
2011).

This fish fanning involves a huge number of farms, from small to large. several small and
large companies, as well as auxiliary services, like major commercial feed industries.
infrastructure for the transportation and marketing of adult fish, fingerling nurseries,
hatcheries that produce fry, and small home feed mills. The development of fish harvesting
teams, mechanical water filling of transport vans, etc. For the impoverished in rural areas,
these activities produced several employment options. Due to its high level of production,
this fish became widely available to the underprivileged and was a significant contributor to
the country's population's diet of animal proteins. But in recent years, the economic gain from
this farming has decreased in part because of rising feed costs, poor management, a shortage
of affordable supplementary feeds, and other socioeconomic limitations (Akter, 2001).
According to reports, Thai pangas farmers are reportedly gradually losing interest in investing
in pangasius farming in the study region (Wahab et al., 2008). It is vital to consider the
sustainability of Thai pangasius (Pangasius sutchi) farming and upgrade the current
pangasius management techniques through institutional initiatives in order to improve the
general conditions of the pangasius farmers (Monir et al., 2011). Thai pangas cultivation has
been expanding over the past ten years, however various reasons have recently had a

7
detrimental impact on it. Thai pangas farming has begun to decline, and farmers have noted
a number of problems, including high input costs and low market prices, improper
management, a lack of affordable supplementary feeds, a lack of quality seed, a lack of
knowledge about quality gametes and broodstock management, among other things.

Thai pangas are one of the most important fish species in Bangladesh which contributes
significantly in the national economy of Bangladesh. The quality sperm is one of the main
pre-requisites for successful breeding of fish. The most of the hatchery owner are not well
known about the importance of sperm quality of any fish species during artificial breeding
program. Due to lack of the knowledge on quality gametes and broodstock management, the
whole aquaculture production will decrease ultimately in near future. To overcome these
problems in present situation, the good quality gamete is required for increasing the
production of aquaculture seeds through artificial breeding program. The main objective of
the proposed study will provide the base-line information about the way of identification of
good quality sperm and to compare the life span of sperm of Thai pangas which will helps to
enhance the quality seed production through artificial breeding in hatchery. The output of this
study will help not only Thai pangas but also helps to commercially important all fish species
in Bangladesh.

8
1.6 Specific objectives of the study:

The aim of this study was to identify the good quality sperm for induced breeding of
commercially important species Thai pangas, Pangasius sutchi in Bangladesh and
secondarily to observe the sperm motility in different time interval. To achieve this principal
goal, the following specific objectives of the proposed study was taken consideration.

Specific Objectives:

➢ To evaluate the effectiveness of NaCl and NaOH on sperm quality of Thai pangas,
Pangasius sutchi in Bangladesh;

➢ To observe the effectiveness of NaCl and NaOH on sperm motility in different time
interval;

➢ To compare the life span of sperm in different time interval; and

➢ To analyze the fertilization rate of eggs using identified quality sperm, and also
determine the hatching rate and survival rate of hatchling.

9
CHAPTER 2

10
 REVIEW OF LITERATURE
It is very important to review the past research works related to the proposed study. Any
quantifiable physical characteristic that is directly related to sperm's ability to fertilize an egg
can be utilized as a benchmark for sperm quality, claim Rurangwa et al. (2004). Since high
motility is a need for fertilization and substantially corresponds with successful fertilization,
motility is the method most frequently employed to quantify sperm quality. Between
spawning seasons in Bangladesh, Hasan et al. (2014) examined the sperm and egg quality in
catla and rui and discovered 90% motile sperms as well as increased sperm concentration.

Considering that males who produce vigorously motile spermatozoa may be able to produce
sperm of higher quality in following generations, Lahnsteiner et al. (1998) recommended the
use of adequate sperm motility assessment as a means of choosing broodstock. In order to
enhance the quality of the broodstock and the welfare of the animals, motility evaluation is
also used to determine the impact of external factors such as stress, diet, husbandry
conditions, and environmental factors.

One of the crucial elements for the activity of fish sperm is salt solution at various
concentrations. There are numerous studies that looked at sperm quality and motility in
various fish species at various salt concentrations. According to Hulak et al. (2008), tap water,
distilled water, 0.2% NaCl, and 0.3% NaCl all stimulated the movement of the common carp
sperm. Like other freshwater fish, the common carp had a relatively brief period of motility
when the temperature was normal. According to Hassan et al. (2013), the length of time that
sperm spent moving forward at various osmolalities of these activating solutions varied
greatly from 58 to 141 s, which is comparable to other carps. A wide variety of hypotonic
solutions can instantly initiate sperm motility, but an ideal osmolality is needed to maintain
motility over time, according to research by Morisawa et al. (1999). When sperm were
suspended in 0.3% NaCl, they exhibited extended periods of motility, indicating that this
activation medium may be used to spawn common carp artificially. A hypo-osmotic shock
can cause mature spermatozoa in freshwater species to become motile.

According to Perchec et al. (1995), Pangasius bocourti spermatozoa had a short motility (57
s) in freshwater but a twice-as-long motility in low saline water. Short motility duration in
freshwater may result from spermatozoa's fast deterioration, as seen in common carp
Cyprinus carpio. According to a recent discovery by Lahnsteiner (2011), Perca fluviatilis

11
(perch) sperm can float for up to two hours in salt water. According to research by Morita et
al. (2004), sperm motility could be activated in freshwater acclimated tilapia at osmolalities
between 0 and 400m Osm/kg with or without electrolytes. Ca2+ was discovered to be
necessary for motility activation.

Sperm in Acipenser transmontanus were found to be pH sensitive, and Ingermann et al.

(2002) reported on this finding. They showed that sperm at high pH (greater than 8.2)
exhibited increased motility when active with water but lower motility at a relatively low pH.
(Less than 7.5). To determine the impact of osmolality on the motility activation of sperm,
common carp sperm were evaluated by comparing the pH of salt solutions with seminal
plasma. Initiation of Cyprinus carpio sperm motility has been documented by Redondo-
Muller et al. (1991) and Perchec-Poupard et al. (1997) in medium with an external pH range
of 6 to 9. According to Ciereszko et al. (2002), Petromyzon marinus sperm motility duration
reduced with increasing pH, while the proportion of motile cells remained constant over the
pH range of 6.0 to 9.0.

Billard and Cosson (1992) claimed that the increase or decrease in the temperature of the
activating solution has a direct influence on sperm motility duration. It is mainly due to the
fact that the energy stock of the fish spermatozoa is limited, and consequently, the increase
in sperm cell activity caused by the rise in the temperature of the activating solution induces
the reduction of sperm motility duration. However, that fall in the temperature of the
activating solution might result in the increase in sperm motility duration due to the reduction
in the cellular metabolism of the spermatozoa (Alavi and Cosson, 2005). The sperm quality
is highly variable and depends on various external factors such as feeding regime, the quality
of the feed and the rearing temperature of the fish (Billard et al., 1995). The contamination of
milt by urine can cause an unexpected activation of spermatozoa which will impair the storage
of milt (Rana, 1995). While Williot et al. (1999) confirmed that Siberian sturgeon, Acipenser
baeri, sperm declines when the temperature is increased from 10 to 17.5°C, Jezierska et al.
(1999) found that sperm are motile for longer at 20°C than at 26 or 30°C in common carp or
30°C in grass carp.

Islam (2002) have observed that induced breeding experiments among 4 doses of PG showed
the best result in the form of fertilization rate (87.89%). Akthteruzzaman et al. (1993), who
recorded a fertilization rate of Pangus 75% at the dose of 3-10 mg PG/kg body weight for
females and 1 mg PG/kg of body weight for male in hapa, among which doses of 2-10 mg

12
PG/kg fish gave the best results. Rahman (2001) found that, the hatching rate of Pangas was
72% in hapa by administration of a single PG dose of 2 and 10 mg PG/kg fish to the female
and 1-2 mg PG/kg fish to the male. Sultana (2011) reported that the average hatching rate of
Pangus 80% when treated with PG doses of 2-12 mg/kg body weight of fish. Successful
induction of P. sutchi using carp pituitary extract was also reported by Chattopadhyay and
Mazumder (2002), with an initial dose of 2–3 mg/kg body weight of fish followed by a
resolving dose of 9.0 or 10.0 mg/kg of body weight for females. Percentage of fertilization
was 77% and 90%, and hatching rates were 87% and 92%, respectively. Akhteruzzaman et
al (1993), who recorded a fertilization rate between 55% and 75% at the doses of 10-18 mg
PG/kg body weight for females and 12 mg PG/kg body weight for the male catfish (Ompok
pabda) in hapa, among which doses of 13-16 mg PG/kg fish gave the best results where the
latency period ranges from 9-10.30 hrs.

According to Santiago et al. (2004), the pangas catfish brood typically had a minimum age of
2 years and weight gains of 2-2.5 kg for females and 2 kg for males. Begum et al. (2001)
described that, the ovulation rates in Heteropneustes fossilis injected with PG at 75 mg/kg
body weight were slightly lower, although they recorded 90% ovulation when the fish were
treated with PG at 100 mg/kg body weight. Using a single intramuscular ovaprim injection at
a rate of 0.3 and 0.5 mg.kg1 body weight for males and females, the induction of ultimate
maturation and ovulation was induced in Heteropneustes fossilis (Godwin Christopher et
al.,2010). According to Godwin Christopher et al. (2010), Heteropneustes fossilis needs a
minimum of two sperm to successfully fertilize an egg. Fertilization success rates ranged from
78 to 93% on average. 98.18% was the greatest fertilization rate ever observed.

Haniffa and Sridhar (2002) successfully spawned Heteropneustes fossilis after giving it a
single intramuscular injection of HCG at doses of 1000, 2000, and 3000 IU/kg body weight.
They observed that Heteropneustes fossilis (Stinging Catfish, Clariidae) administered with
the synthetic hormone Ovaprim at a dose rate of 0.3 ml/kg-1 body weight experienced a 70%
fertilization rate. At the end of a 14-day indoor experimental period, lower fry survival of
68.2% and greater fry survival of 85% were documented in this study. Ovaprim, Ovatide, and
WOVA-FH were employed by Karal Marx and Ramanuj Chakraborty (2007) to stimulate
oocyte maturation and ovulation in Heteropneustes fossilis at doses of 0.5, 1.0, 1.5, and 2.0
ml/kg body weight. The eggs were stripped and in vitro fertilization was carried out after 10
to 13 hours of injection. The highest hatching rate was achieved by Ovaprim (94.67%),
followed by Ovatide (90.33%) and WOVA-FH (77.33%).

13
In order to stimulate oocyte maturation and ovulation, Nayak et al. (2001) administered 17-
hydroxy progesterone (17-P) into the female stinging catfish (H. fossilis) with or without
priming with PG homogenate. While 8 g/g body weight dosages were used to achieve this,
administration of 8 mg/kg body weight PG homogenate with 5 g/g body weight of 17-P
resulted in greater egg production. Most of the eggs that were released between 14 and 18
hours after the administration were fertilized and hatched normally. Olufeagba et al. (2015)
investigated Heteropneustes fossilis induced breeding, embryogenic timing, and larval
development. A sexually mature female was given an intramuscular injection of a dry carp
pituitary gland suspension weighing 4 mg/kg. After an eight-hour latency period, the man
was slain while the female was stripped in order to remove the testicles that would have been
macerated to fertilize the eggs. The various developmental stages were shot while the embryo
was incubated at 27°C. Hatching began 23.53 hours after fertilization, with a 97.5%
hatchability rate. The average survival rate was 75%.

In laboratory aquaria and hapas fixed in ponds, Parameswaran et al. (1971) were successful
using preliminary doses of 2 to 3 mg and a final dose of 8 to 22 mg PG/kg body weight of the
fish that were administered 4 to 6 hours after the first injection for the female and a single
injection of 3 to 5 mg PG/kg of the males at the time of the second injection to the female
Shingi. The spawning habits of the air-breathing catfish Heteropneutes fossilis were studied
by Thakur et al. in 1977. They discovered that the spawning activity began between 6 and 10
hours after the pituitary injection. In separate trials, the activity lasted between 2 and 6 hours.
Intermittent mating behaviors were seen during this time, with the average rate of mating
being once every 2 to 3 minutes in the early stages and once every 5 to 10 minutes or longer
in the latter ones.

This research review’s purpose is to help the reader understand different aspects posed by the
research on sperm quality and sperm motility of Thai pangas, Pangasius sutchi in Bangladesh.
Along with this, this research also helps to analyze the fertilization rate of eggs using
identified quality sperm, and also determine the hatching rate and survival rate of hatchling.

14
CHAPTER 3

15
 MATERIALS AND METHODS
3.1 Study Site

The present study was conducted between April 2022 and July 2022 at Chanchal Matshya
Hatchery which is located approximately 15 km northeast of the Patuakhali District. The
Hatchery which covers 7 acres of land has the following facilities; such as indoor hatchery
complex with 10 hatching tanks (7 ft x 5 ft each), 5 breeding tanks (10 ft x 5 ft each), 10
earthen nursery ponds (4 ft x 3 ft x 3.5 ft each), two brood stock ponds (6 ft x 5 ft x 5 ft each),
and a water reservoir (10000 L) etc.

3.2 Sperm Collection

Ready-to-spawn broods were collected from the ponds and conditioned for 12 hours in the
hatchery tanks. Males of P. sutchi injected with 2.0 mg kg-1 PG in order to increase the volume
of milt collected by striping. After 6 hrs from PG injection, the males were captured from the
tank using a scoop net and were laid on foam to wipe the urogenital pore. Gentle pressure
was applied through the abdomen to remove urine, water, gut exudates and mucus to avoid
contamination. Sperm were collected in glass vials by abdominal pressure and the vials were
immediately placed at 4 °C. Ejaculated sperm volume was determined by the measuring
pipette and expressed as microliter (µl).

3.3 Experimental Design

A total of 20 females and 8 males with total body weight of 55 kg and 20 kg were allocated
into three treatments in a complete Randomized Design and labeled as T1 and T2 where T1
and T2 means the effectiveness of NaCl and NaOH on sperm quality. Each treatment has 3
replications and labeled as R1, R2 and R3 where R1, R2 and R3 represents the % of NaCl
and NaOH solutions. Sperm of males of each replication were kept separately, and both males
and females under treatments, T1 and T2 were treated with PG hormone at single dose for
male and double doses for female are presented in Table 3.1 and Table. 3.2.

16
Table 3.1. Effectiveness of NaCl and NaOH on sperm quality in different % of these solutions

Control Treatment 1 (T1) Treatment 2 (T2)

NaCl NaOH
R1 R2 R3 R1 R2 R3
Normal Tape Water 1% NaCl 3% NaCl 5% NaCl 1% NaOH 3% NaOH 5% NaOH

Table 3.2. Single dose of PG in each male and double doses of PG in each female Thai
pangas, Pangasius sutchi.

Hormone Doses Male Female

PG 1st 2.0 mg kg-1 4.0 mg kg-1
2nd - 2.0 mg kg-1

3.4 Sperm Quality Assessment

The quality of stripped milt sample was evaluated after inducing agent’s treatments. The
physico-chemical parameters of milt like pH and concentration of sperm were determined
using glass slide. The sperm of different species was observed under the biological
microscope (BEL Engineering, Binocular, achromatic optics, LED) and also took picture and
then analyzed the morphology of sperm.

3.5 Motility Assessment

The milt of Thai pangas was evaluated for sperm yield kg-1 body weight, motility, pH,
spermatocrit percentage and then sperm count. Spermatozoa motility assessment was carried
out by diluting milt with sterile water (1:100) at room temperature (28 °C) on petridish,
observed immediately under biological microscope (100 X) with a camera. Estimation of
spermatozoa motility started immediately (approximately 10 s) after dilution and the
movement was observed till 0 min, 15 min, 30 min, 60 min and 90 min. The motility was
recorded in a computer.

17
The dilution of sperm prepared by using following procedure and then the number of sperm
counted by the following equation (each treatment has at least 3 replicates).

0.1 ml sperm + Additional 10 ml working solution (100 times dilution)

Kept 0.2 ml on slides

Observation under microscope

Counting of motile sperm

Total number of sperm in 0.1 ml = No. of sperm in 0.2 ml working solution × 50

3.6 Assessment of Life Span of Sperm

The life span of Thai pangas sperm was then compared in different time interval (0 min, 15
min, 30 min, 60 min and 90 min) and assess the survivality of highest duration of sperm in
different species.

Fig. 3.1. Flow diagram of PG hormone administrated on both male and female Thai pangas,
Pangasius sutchi.

18
Fig. 3.2. Flow diagram of sperm quality assessment under biological microscope

3.7 Analysis of Ovulation Rate (%), Fertilization Rate (%) of Eggs, Hatching Rate (%)
and Survival Rate (%)

The eggs were collected from female brood fish by stripping method. Then the eggs were
fertilized with sperm at different time intervals (0 min, 15 min, 30 min, 60 min and 90 min)
and analyzed fertilization rate after 3 hr. The fertilized eggs were incubated at room
temperature until hatching. The following parameters like ovulation and fertilization rate (%)
of eggs, hatching and survival rates (%) of larvae were determined by using the following
formula.

Ovulation rate (%) = (Number of fish ovulated/ Total number of fishes injected) × 100

Fertilization rate (%) = (Number of fertilized eggs or embryos/ Total number of eggs) × 100

19
Hatching rate (%) = (Number of hatched larvae/ Total number of fertilized eggs) × 100

Survival rate (%) = (Number of survival larvae after 3 and 5 days/ Total number of hatched
larvae) × 100

3.8 Statistical analysis

All data was expressed as mean ± SD (standard deviation). The motility, survivality and
mortality of sperm was expressed as percentages before statistical evaluation. Analysis of
variance and Duncan’s new multiple range tests used to test the difference among treatments
at 5% level of significance. All statistics were carried out using SPSS for Window version
16.0.

20
CHAPTER 4

21
 RESULTS
4.1 Effects of NaCl and NaOH on Sperm Quality

Swimming media (either NaCl or NaOH solutions) led to high percentages of motile
spermatozoa (90-100 %) and, in comparison to normal tape water, reduced damages to
flagellum. Sperm quality in NaCl solution with 1%, 3% and 5% concentration and its effects
were recorded as 17234 ± 1013, 21617 ± 1419 and 24344 ± 1734 in T1R1, T1R2, T1R3. The
highest number of sperm counted as 24344 ± 1734 in T1R3 and lowest number of sperm
counted as 17234 ± 1013 in T1R1 (Table 4.1). Effects of NaOH solution with 1%, 3% and
5% concentration on sperm quality were recorded as 7219 ± 879, 8503 ± 475 and 10373 ±
376 in T2R1, T2R2, T2R3. The highest number of sperm counted as 10373 ± 376 in T2R3
and lowest number of sperm counted as 7219 ± 879 in T2R1 (Table 4.1).

4.2 Effects of NaCl and NaOH on Sperm Motility

Different swimming media affects the sperm quality differently and its effects on sperm
motility were also different. In normal tape water, after immediate ejaculation the sperm
number were 20989 ± 1179. But after 15 min, 30 min, 60 min and 90 min, the sperm number
reduced to 14701 ± 323, 5259 ± 179, 422 ± 11 and 63± 2. In NaCl, after immediate ejaculation
the sperm number were 24344 ± 1734. But after 15 min, 30 min, 60 min and 90 min, the
sperm number reduced to 18053 ± 453, 7208 ± 87, 1217 ± 124 and 144± 7. In NaOH, after
immediate ejaculation the sperm number were 10373 ± 376. But after 15 min, 30 min, 60 min
and 90 min, the sperm number reduced to 6337 ± 437, 2033 ± 94, 103 ± 19 and 0 (Fig. 4.1
and Table 4.2).

4.3 Ovulation Rate (%) of Pangasius sutchi and Fertilization Rate (%) of their Eggs
The highest ovulation rate was found to be 84 ± 3.9% and 91 ± 2.2% in T1 and T2,
respectively (Table 4.3). There was no significant different (p > 0.05) was found in T1 and
T2. The fertilization rates were recorded as 65 ± 1.2% and 70 ± 0.8% in T1 and T2,
respectively (Table 4.3). There was no significant different (p > 0.05) was found in T1 and
T2.

22
Table 4.1. Evaluation of the effectiveness of NaCl and NaOH on sperm quality at different
concentration.

Treatment 1 (T1) Treatment 2 (T2)

R1 R2 R3 R1 R2 R3
Control

Normal Tape Water 1% NaCl 3% NaCl 5% NaCl 1% NaOH 3% NaOH 5% NaOH

20989 ± 1179 17234 ± 21617 ± 24344 ± 7219 ± 8503 ± 10373 ±

1013 1419 1734 879 475 376

Table 4.2. Evaluation of the effectiveness of NaCl and NaOH on sperm motility in different
time interval

0 min 15 min 30 min 60 min 90 min

Normal 20989 ± 14701 ± 323 5259 ± 179 422 ± 11 63 ± 2
Tape Water 1179 (70%) (25%) (2%) (0.3%)
(100%)
5% NaCl 24344 ± 18053 ± 453 7208 ± 87 1217 ± 124 144 ± 7
1734 (75%) (30%) (5%) (0.6%)
(100%)
5% NaOH 10373 ± 376 6337 ± 437 2033 ± 94 103 ± 19 Nil
(100%) (60%) (20%) (1%)

4.4 Hatching Rate (%) and Survival Rate (%) of P. sutchi Larvae
The hatching rate of Thai pangas was found 94 ± 1% and 96 ± 0.8% in T1 and T2, respectively
(Table 4.3). There was no significant different (p > 0.05) was found in T1 and T2. The
survival rate of hatchlings was observed in both treatments at different time intervals 24 h, 48
h and 72 h of Thai pangas, P. sutchi. The highest (90%) and lowest (70%) survival of
hatchlings was found at 24 h and 72 h in Freshwater media, respectively whereas highest
(92%) and lowest (72%) survival of hatchlings was found at 24 h and 72 h in 5% NaCl media,
respectively (Table 4.4). The survival rate was significantly different (p < 0.05) in each

23
treatment in different time intervals. On the other hand, no significant different (p > 0.05) was
found in T1 and T2 in same time intervals.

Table 4.3. Evaluation of the average ovulation rate (%), fertilization rate (%), and hatching
rate (%) of Pangasius sutchi broods, fertilized eggs, and their hatched larvae, respectively
after PG dose administered.

Parameters Normal Tape Water 5% NaCl

Ovulation rate (%) 84 ± 3.9 % 91 ± 2.2 %

Fertilization rate (%) 65 ± 1.2 % 70 ± 0.8 %

Hatching rate (%) 94 ± 1.0 % 96 ± 0.8 %

Table 4.4. Effect of PG hormone on the survival rate (%) of Thai pangas, Pangasius sutchi
in different time intervals 24 h, 48 h and 72 h.

Survival rate (%)

Time interval

Normal Tape Water 5% NaCl

24 h 72 ± 0.7% 74 ± 0.68%

48 h 68 ± 0.62% 70 ± 0.64%

72 h 66 ± 0.54% 68 ± 0.56%

24
25
CHAPTER 5

26
 DISCUSSION
Thai pangas, P. sutchi is an important and demandable species in all kinds of Bangladeshi
people due to Its good taste and deliciousness and also it contains high nutritional value. The
recruitment of wild fish as well as the controlled production in aquaculture are biological
events strongly linked to the reproductive success and in particular, with the fertilization of
mature oocytes and sperm quality. It is reasonable to imagine that fertilization success will
depend on gamete quality and, focusing in males, it makes sense to define and understand
what gamete (sperm) quality means. From a biological point of view, sperm quality could be
defined as the ability of the spermatozoa to exploit their swimming ability for reaching and
fertilize the oocyte (Fauvel et al. 2010). The present study was conducted to evaluate the good
quality sperm of commonly culturable species Thai pangas in Bangladesh and secondarily to
observe the sperm motility in different time interval.

The results of the present study are discussed below comparing with the findings of other
researchers in the relevant fields. Hasan et al. (2014) worked on sperm and egg quality in
catla and rui between spawning seasons in Bangladesh and found 90% motile sperms as well
as higher sperm concentration. In present study, similar result found as different swimming
media at different concentration (either NaCl or NaOH solutions) led to high percentages of
motile spermatozoa (90-100%) in comparison to freshwater.

Hulak et al. (2008) described that motility of the common carp sperm was triggered by tap
water, distilled water, 0.2% NaCl and 0.3% NaCl. Motility duration of common carp was
very short like other freshwater fish at ambient temperature. But in present study, Thai pangas
sperm was triggered by freshwater, 5% NaCl and 5% NaOH. Motility duration was better in
5% NaCl than other two media. According to Morisawa et al. (1999), sperm had greater
motility duration when suspended with 0.3% NaCl, indicating that this method could be used
to activate common carp during artificial spawning. Similar findings were made in the current
investigation, which concluded that 5% NaCl solution was the best activation medium for
Thai pangas.

According to Perchec et al. (1995), Pangasius bocourti spermatozoa had a brief motility in
freshwater (57 s), while their motility in low saline water was twice as long. Short motility
duration in freshwater may result from spermatozoa's fast deterioration, as seen in common
carp Cyprinus carpio. According to a recent discovery by Lahnsteiner (2011), Perca

27
fluviatilis (perch) sperm can float for up to two hours in salt water. Similar kind of result
found in the present study that sperm motility was short in freshwater but long in low saline
water.

The present study suggested that both NaCl and NaOH solutions helps to increase sperm
motility of Thai pangas. The maximum duration of sperm motility was demonstrated by
Morisawa et al. (1983) in rainbow trout (20 s) and masu salmon (15–25 s) at a sodium
concentration between 0 and 150m molkg-1 (300m osmolkg-1). Beata et al. (2018) found that
independent of sodium and potassium contents, sperm motility was high immediately upon
dilution (0 h) (range 81.5-93.6%) in all artificial seminal plasma (ASP) of common carp
Cyprinus carpio L. variants. Sperm motility considerably decreased over time; after 72 hours
of preservation, the ASP supplemented with 150 mM KCl without salt addition had the lowest
sperm motility (12.8%). A similar pattern was seen in the increasing sperm motility. More or
less similar result was found in the present research as sperm motility was high immediately
upon dilution (0 min) and sperm motility considerably decreased over time; after 90 min 5%
NaCl containing solution had the lowest sperm motility (0.6%). There was no survival sperm
was observed after 90 min from ejaculation of sperm at 5% NaOH solution.

Sperm motility was evaluated at various salt solution concentrations (% NaCl solution) in the
present study. In case of sperm motility, solution the total motile sperm was counted as 17234
± 1013 at 1% NaCl solution whereas it increased to 21617 ± 1419 and 24344 ± 1734 in 3%
and 5% NaCl solutions, respectively. Sperm motility was more or less steady and appeared
fully active at 5% NaCl solution. The result of 5% NaCl solution was significantly different
than 1% NaCl solution. Nafia et al. (2020) found that sperm motility was more or less steady
and appeared fully active at 0.4% NaCl. They also found that the sperm motility in a 0.1%
NaCl solution was 80.0±3.5%, but it increased to 93.4±2.30 and 87.40±4.9 in 0.4% and 0.5%
NaCl solutions, respectively. The result of the present study on sperm motility is the opposite
with Nafia et al. (2020).

After conditioning of brood fishes, the weight of each fish was taken for the preparation of
PG dose. It was observed that, the broods of Thai pangas were generally minimum age of 2
years and weight of male and female were minimum 2 and 2.8 kg of each fish. This value is
more or less similar to Santiago et al. (2004) who mentioned that the cat fish brood of Pangas
were generally minimum age of 2 years and weight were gained female 2-2.5 kg and male 2
kg.

28
The result of the present study was revealed that the fish respond well in induced breeding
using single dose of PG 4.0 mg/kg and 2.0 mg/kg body weight of female and male,
respectively Akhteruzzaman et al. (1993) achieved success by using a single dose of 10-l8
mg PG/kg body weight for females and 12 mg PG/kg body weight for the males of catfish.
Among various PG doses which were tested and gave the best doses ranges from 13-16 mg
PG/kg fish. Parameswaran et al. (1971) also achieved success by using a preliminary dose of
2 to 3 mg and a final dose of 8 to 22 mg PG/kg body weight of the fish administered 4 to 6 h
after the first injection for the female and a single injection of 3 to 5 mg PG/kg of the males,
at the time of second injection to the female shingi both in laboratory aquaria and hapas fixed
in ponds. The result of the present study revealed that the fish respond to ovulate by a single
PG dose 4 mg/kg body weight of fish to female. The male was found to be responding by a
single dose of PG i.e., 2.0 mg/kg fish which is higher than previously mentioned Adebiyi et
al. (2013) that was 0.03-0.5 ml/kg female and 0.4-0.6 ml/ kg males and lower than of 0.7
ml/kg for female and 0.5 ml/kg for male for butter catfish Ompok bimaculatus (Raizada et al.
2013). This high dose response may be due to the influences the factors like temperature and
quality of water as well as the manipulation of hormone dose like using only a single dose of
hormone to avoid more chances for getting injured during handling of the fish.

The outcome of the current investigation revealed variations between the two methods of
ovulation induction. In 5% NaCl solution, the average ovulation rate was highest (91±2.2%),
while normal tap water had the lowest value (84±3.9%). The results showed that the 5% NaCl
solution was better than regular tap water. Ovulation period is presumably the reason why the
ovulation rate was lower in normal tape water than at 5% NaCl solution. The ovulation rates
in Ompok pabda fed with PG at 75 mg/kg body weight were slightly lower, while they
reported 90% ovulation when the fish were treated with PG at 100 mg/kg body weight,
according to research by Begum et al. (2001). However, the fertilization rate with ovaprim
(at 0.06 ml/100 gm for males and 0.12 ml/100 gm for females) was lower (on average
65±0.8%) than in the earlier publication by Chaturvedi et al. (2015).

The highest fertilization rate of Thai pangas was reported in the current study to be 70% for
a dose of 2-4 mg PG/kg, and the lowest rate was discovered to be 65%. Islam (2002) noted
that the fertilization rate (87.89%) of induced breeding tests using 4 doses of PG demonstrated
the greatest outcome. The results of Akthteruzzaman et al. (1993) who recorded a fertilization
rate of Pangas 75% at the dose of 3-10 mg PG/kg of body weight for females and 1 mg PG/kg
of body weight for males in hapa, among which doses of 2-10 mg PG/kg fish gave the best

29
results, are consistent with the current findings of fertilization rate. Additionally, with 5%
NaCl solution, the fertilization rate was shown to be 5% higher at 65±1.2% and 70±0.8%.
Additionally, the fertilization rate was observed to be 5% higher at 5% NaCl solution as
65±1.2% and 70±0.8% compared to regular tap water. Chattopadhyay and Mazumder (2002)
also reported successful induction of P. sutchi using carp pituitary extract, with an initial dose
of 2-3 mg/kg body weight of fish followed by a resolving dose of 9.0 or 10.0 mg/kg body
weight for females. Hatching rates were 87% and 92%, whereas fertilization percentages were
77% and 90%, respectively. Similar findings were made in this study, where the greatest
fertilization rate for Thai pangas was found to be 70% and the lowest rate to be 65%.

Similar result was observed by Haniffa and Sridhar (2002) for induced breeding of stinging
catfish H. fossilis, where Ovaprim, another synthetic hormone, was used as inducing agent
and was found 70% fertilization of H. fossilis whereas optimal dose was 0.3 ml/kg-1 body
weight. Akhteruzzaman et al. (1993) reported that highest fertilization rate between 55% and
75% was found at the doses of 10-18 mg PG/kg body weight for females and 12 mg PG/kg
body weight for the male catfish (Ompok pabda) in hapa. Godwin Christopher et al. (2010)
reported that the minimum number of sperms required for optimal fertilization success in H.
fossilis and also recorded 78–93% fertilization success of his study. The fertilization rate was
lower than the other study due to the fluctuation of temperature and also for reduced
precipitation.

During the experimentation with dose optimization of PG the hatching rate were found to be
94 ± 1.0% and 96 ± 0.8%in normal tape water and 5% NaCl solution, respectively. The
highest hatching rate was recorded as 96 ± 0.8% in 5% NaCl solution and the lowest hatching
rate was recorded as 94 ± 1.0% in normal tape water. More and less similar findings were
recorded by Raizada et al. (2013), who reported that the latency period for spawning of O.
bimaculatus was 7–8 h. at temperature 27 ± 0.5 °C and fertilization and hatching rates were
found in the range of 75–90% and 80–90% respectively in flow-through system. Rahman
(2001) discovered that giving females a single PG dose of 14 and 16 mg/kg fish and males a
single dose of 12 fish resulted in a 95% hatching rate in hapa. In a related study, Nayak et al.
(2001) reported that Ovaprim treatment of H. fossilis at 27 °C resulted in a hatching duration
of 10–12 h and a greater hatching rate of 96% using Ovaprim at the rate of 0.4 ml/kg body
weight. In a different study, Roy and Pal (1986) examined H. fossilis (Bloch.) induced
breeding in laboratory aquaria and discovered that over 93% of the released eggs hatched in
21–24 hours at a temperature of 24-31°C. Vijaykumar (2009) also investigated on some other

30
species of Ompok and noticed a poor success in Ompok malabaricus. Simultaneously with a
similar study certain rate of the hatchling is found in Ompok pabda (Chakraborty et al. 2007,
2008). Karal Marx and Ramanuj Chakraborty (2007) also obtained results that were quite
comparable. Overall, this study's hatching rate may be higher than that of prior studies.

Additionally, the survival rate for each treatment at various time points was computed (24 h,
48 h and 72 h). In regular tap water, hatchling survival rates were 72%, 68%, and 66% at 24,
48, and 72 hours, respectively. In contrast, hatchling survival rates in a 5% NaCl solution
were 74%, 70%, and 68% at 24, 48, and 72 hours, respectively. After 24 hours, the survival
rate was 74 0.68%; after 72 hours, it substantially dropped to 66 0.54%. According to the
study, the survival rate was dropping daily. The data also revealed that in all cases, survival
was over 2% higher in the 5% NaCl solution than in the regular tap water. At 24 hours after
hatching compared to 72 hours after hatching, the survival rate was about 10% higher in both
treatments. The survival rate at that time was roughly consistent, approximately 68. At the
conclusion of the 14-day indoor trial period, 2% survival was discovered. Islam (2011)
observed the highest survival rate as 60.40 ± 0.59% in June and the lowest rate as 42.76 ±
0.55% in August, 2011 in O. pabda. The survival rate of P. sutchi in the present study was
also higher with PG injected fish. Ovaprim-treated fish outlived other hormonal materials
used to develop the stinging catfish H. fossilis, according to Haniffa and Sridhar (2002). It
can result in natural death or it might have the impact of an abrupt temperature change.
Induced breeding and larval rearing of the stinging catfish, H. fossilis, by Chaturvedi et al.
(2015) and Olufeagba et al. (2015) yielded similar results.

31
CHAPTER 6

32
 SUMMARY AND CONCLUSION
Thai pangas, P. sutchi is one of the most important commercial catfish in Bangladesh. It has
been drawing the attention of fish farmers in Bangladesh day by day due to its high market
demands and profitable culture. Studies on induced breeding of P. sutchi were carried out by
several researchers in Bangladesh. This study has conducted to acquire experience on the
quality of sperm, the motility of sperm, breeding performance, fertilization rate, hatching rate
and survival rate. The whole works was carried out in Chanchal Matshya Hatchery in Bauphal
at Patuakhali District where all necessary facilities were available.

The outcome of the current investigation demonstrated substantial variations between the two
therapies' efficacy in assessing sperm quality and motility. The effects of the sodium chloride
solution included the largest number of sperm counted at 24344±1734 in T1R3 and the lowest
number at 17234±1013 in T1R1. In the other instance, the NaOH solution's effects included
the highest sperm count of 10373±376 in T2R3 and the lowest sperm count of 7219±879 in
T2R1. On the other hand, a significant (p 0.05) decreasing trend was seen in the sperm
motility at 5% NaCl and 5% NaOH concentrations in different time periods, namely 0 min,
15 min, 30 min, 60 min, and 90 min following ejaculation. On the other hand, a significant (p
0.05) declining tendency was seen in all treatments for sperm motility at 5% NaCl and 5%
NaOH concentrations in different time periods, including 0 min, 15 min, 30 min, 60 min, and
90 min following ejaculation. The results of the current study revealed that 5% NaCl solution
provided the highest quality and motility of sperm in Thai Pangas, with regular running water
and NaOH solution coming in second and third.

For the effectiveness of two treatments in between normal running water and 5% NaCl
solution, females and male fishes were treated with single dose which administrated 4.0 ml/kg
and 2.0 ml/kg, respectively. The highest ovulation rate was recorded 91% in 5% NaCl
solution whereas the lowest rate was 84% in normal running water, fertilization rate was
recorded 65% and 70% in normal running water and 5% NaCl solution, respectively. Not
only the highest (96 ± 0.8%) and lowest (94 ± 1.0%) hatching rate was also recorded in 5%
NaCl solution and normal running water, respectively but also the highest survival was also
found in 5% NaCl solution after 72 h rearing of Thai pangas hatchling. On the basis of Thai
pangas' ovulation rate, egg fertilization rate, hatching rate, and hatchling survival rate, the
study concluded that a 5% NaCl solution was preferable to regular running water. However,
it is difficult to pinpoint the reason for such differing results because a number of factors

33
affect the biological experiment particularly involving hormones. Several major factors that
may have bearing on the result are: (a) age and physiological state of fish, (b) the seasonal
variation, (c) environmental factors such as water temperature, dissolved oxygen etc. and (d)
source of fish (wild or farmed).

Although the experiment was conducted with limited facility, the consequence is hopeful and
would serve up to the fish farmer and hatchery owner. The findings of this study provide the
basis for the potential to be a guide for further researches to evaluate the good quality sperm
of Thai pangas, Pangasius sutchi in Bangladesh. In this regard, it is suggested that the
following points must be addressed in future studies:

➢ Further study is needed to find out the effect of swimming media (either NaCl or
NaOH solutions), which is used to evaluate the good quality sperm not only of Thai
pangas, Pangasius sutchi but also other related fish species.
➢ Should also carry out further experiment and trial at hatchery on related species
perhaps other fishes under different area of Bangladesh.

34
CHAPTER 7

35
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