effects of steroid on artifical propagation of striped catfish (pangasianodon hypophthalmus)

The objective of my study is to find out the effects of steroid 17,20P on artificial propagation of striped catfish Pangasianodon hypophthalmus.. 17 4.1 The experiment 1: Stimulate strip

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

EFFECTS OF STEROID ON ARTIFICAL PROPAGATION

OF STRIPED CATFISH

(Pangasianodon hypophthalmus)

By

NGUYEN MINH LINL

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture

Can Tho, December 2012

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

EFFECTS OF STEROID ON ARTIFICAL PROPAGATION

OF STRIPED CATFISH

(Pangasianodon hypophthalmus)

By

NGUYEN MINH LINL

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture

Supervisors

Dr BUI MINH TAM

Dr PHAM THANH LIEM

Can Tho, December 2012

i

Acknowledgements

I wish to express my deep appreciation and sincere gratitude to Dr Bui Minh Tam and Dr Pham Thanh Liem for their guidance, assistance and advice have been a great help in my thesis

I am thankful to the support from the staff and students in the hatchery of College of Aquaculture and Fishery, Can Tho University Especially, I would like to thank Mr Nguyen Hong Quyet Thang for his contributions

in solving technical problems Moreover, I would like thank the staffs of Hung Vuong Ba Tri Company for their kind supports during my study

Finally, I wish to thank my parents for their support and encouragement throughout my study

Abstract

Striped catfish (Pangasianodon hypophthalmus) – known more universally

by the Vietnamese name 'Tra' is an economically important species of fresh water fish in the Mekong Delta in Vietnam, which constitutes an important food resource Artificial propagation technology for Tra catfish has been recently developed along the main branches of the Mekong River where more than 60% of the local human population participate in fishing or aquaculture Extensive support for catfish culture in general and that of Tra

(Pangasianodon hypophthalmus) in particular, has been provided by the

Vietnamese government to increase both the scale of production and to develop international export markets

The objective of my study is to find out the effects of steroid (17,20P) on

artificial propagation of striped catfish (Pangasianodon hypophthalmus) The

result showed that the application of HCG 1500UI/kg of female in the preliminary dose and of 17,20P at levels of 5-6 mg/kg of females in the decisive dose gave the ovulation rate of 28% Also, the use of preliminary dose by pituitary gland at 4 mg/kg of females and the decisive dose by 17,20P at levels of 5-6 mg/kg of female gave the ovulation rate of 51% and hatching rate of 31% Using HCG 1200UI/kg in the preliminary dose and combination of 500UI HCG and 3, 4, 5 mg/kg of 17,20P in decisive dose gave the ovulation rate 20%, 40%, 15% and hatching rate 63%, 74%, 58%, respectively Thus, the application of 17,20P in striped catfish breeding would be convenience for fish breeding farmers that they can avoid the storage of protein or peptide hormones in restricted conditions (low temperature) such as gonadotropin and gonadotropin releasing hormones

iii

Table of Contents

Acknowledgement i

Abstract ii

Table of contents iii

List of table iv

List of figures v

List of abbreviations vi

Chapter I: Introduction 1

1.1 Background of the study 1

1.2 Objective of the study 1

1.3 contents of study 2

Chapter II: Literature review 3

2.1 Biological characteristics of striped catfish 3

2.1.1 Morphology and distribution 3

2.1.2 Reproduction 5

2.1.2.1 Hormone control oocyte maturation 5

2.1.2.2 Development of Oocytes 7

2.1.2.3 Final oocyte maturation and ovulation 8

2.2 Maturation inducing steroid 17,20P (17α, 20β-dihydroxy-4-pregne-3-ones) 9 Chapter III: Methodology 11

3.1 Time and lace 11

3.2 Materials 11

3.3 Experimental study 12

3.3.1 Experiment 1 12

3.3.2 Experiment 2 12

3.4 Broodstocks selection 13

3.5 Removal of eggs and milt, fertilization 13

3.6 Data analysis 15

3.7 Method of collecting and analyzing data 15

Chapter IV: Results and discussion 17

4.1 The experiment 1: Stimulate striped catfish spawning using HCG and pituitary gland in the priming dose and 17,20P (17α, 20β-dihydroxy-4-pregne-3-ones) in the decisive dose 17

4.2 The experiment 2: stimulate catfish spawning using HCG in the priming dose and combination between HCG and 17,20P (17α, 20β-dihydroxy-4-pregne-3-ones) in the decisive dose 20

4.3 The effectiveness comparison of using 17,20P and Ovaprim, HCG in artificial propagation 22

4.4 Embryonic development 22

Chapter IV: Conclusion and recommendation 25

5.1 Conclusions 25

5.2 Recommendation 25

References 26

Appendices 29

List of tables

Table 3.1: Stimulating striped catfish spawning by using HCG or PG in priming dose and 17,20P in decisive dose 12Table 3.2: The use preliminary dose by HCG and decisive dose by combination

of HCG and 17,20P 12

Table 4.1: The result of experiment 1 at hatchery of College of Aquaculture and

Fishery 16

Table 4.2: The result of experiment 1 at the Hung Vuong Ba Tri Company 17

Table 4.3: The result of experiment 2 at hatchery of College of Aquaculture and Fishery 19 Table 4.3: Process of embryonic development 23

v

List of Figures

Figure 2.1Striped catfish (Pangasianodon hypophthalmus) 3

Figure 2.2 Diagram of hormone control oocyte maturation 6Figure 4.1 Embryos development of striped catfish 24

CHAPTER I INTRODUCTION 1.1 Background of the study

Aquaculture of striped catfish, Pangasianodon hypophthalamus (Sauvage, 1878),

in the Mekong Delta of Vietnam, is considered as one of the fastest growing sectors of the food production It achieved a production level of 1.195 million tons in 2011, the average yield is about 400 tones/ha/crop and over 90% of the product is processed and exported to more than 130 countries In 2011, sector generated about 1.8 billion US$ of export income The catfish farming in the Mekong Delta also provides a significant employment in the area (http://www.fistenet.gov.vn, 2012)

Strong development of farming has a very high demand of artificial seeds Beside pituitary gland and HCG, scientists have found several active ingredients to stimulate the production of reproductive fish, such as sGnRHa (LHRH, Ovaprim) Currently, most of hatcheries often use 4 types of active ingredients to stimulate the production They are pituitary gland, HCG, LHRH + Domperidon and Ovaprim These compounds are derived proteins which are instable quality and expensive, difficult to manage (decomposed at high temperature and infection), high price and instability of quality Most of these hormones make the broodstock secrete all gonadotrophic Therefore, broodstock becomes exhausted and need a long time to recover Using steroid hormone can limit these problems because of some advantages as following:

 The steroid hormones can stimulate fish spawn earlier

 The steroid hormone is not decomposed by bacteria and at high

temperature The excess use of drugs can be preserved and saved

The research on "Effects of steroid on artificial propagation of striped catfish

(Pangasianodon hypophthalmus)" was conducting contribute to aquaculture

development Particularly, it plays an important role in creating of artificial seed

sources and rearing fish species that have high economic values

1.2 Objective of the study

The overall objective is to find out effects of steroid on artificial propagation of

striped catfish (Pangasianodon hypophthalmus)

1.3 Contents of the study

 To stimulate catfish spawning using pituitary gland (4mg/kg) or HCG (1500UI/kg) in the priming dose and 17,20P at different dosages (5, 6 mg/kg of females) in the decisive dose

 To stimulate striped catfish spawning by using HCG in the preliminary dose and combination of 500UI/kg HCG and 3, 4, 5 mg/kg of 17,20P in the decisive dose

CHAPTER II LITERATURE REVIEW 2.1 Biological characteristics of striped catfish

2.1.1 Morphology and distribution

Morphological classification of striped catfish is as follows:

Species Pangasianodon hypophthalmus (Sauvage, 1878)

Figure 2.1: Striped catfish Pangasianodon hypophthalmus ( Pham Van Khanh,

1996)

Striped catfish, Pangasianodon hypophthalmus (Sauvage 1878), has synonyms as Pangasius hypophthalmus (Sauvage 1878).Trey pra thom in Khmer, Pa souay kheo in Lao, Pla sawai in Thai, Ca Tra in Vietnamese The species is relatively

large in sizes, it can grows up to a maximal weight of 44 kg in the wild (Fishbase, 2004)

Striped catfish have air-breathing organs and obligate air breathers (Cacot, 1999) Therefore, they tolerate poor water quality and can be stocked at high densities The species is omnivorous; therefore they can accept a wide variety of feed, e.g trash fish, pellets, home-made feed formulated from agronomy and fishery byproducts, aquatic plants and even animal and human wastes (Rainboth, 1996; Roberts and Vidthayanon, 1991)

Sexual maturation of striped catfish takes more than 3 years in captivity (Catot, 1999) with a weight of 3-4 kg, corresponded with a total length of 54 cm (Touch, 2000) Striped catfish is a prolific spanner, egg production increases with increasing size until a weight of around 10kg, and some are capable of spawning twice a year (Cacot, 1999)

Striped catfish inhabits large rivers Its natural distribution range is limited from the Chao Phraya River to the Mekong River basin (Roberts and Vidthayanon, 1991) The species is also found in the Ayeyawady Basin of Myanmar In the lower Mekong basin, there may be two stocks of striped catfish, the southern stock inhabiting the vicinity of Mekong River in Cambodia and Vietnam, and the northern stock above the Khone falls, in Laos and Thailand (Poulsen and Valbo-Jørgensen, 2011)

Spawning grounds of the southern stock were identified in Mekong River from the Stung Treng Province in Cambodia, concentrated from Prek Kampi to Koh Rongie, Kratie Province, in Cambodia (Touch 2000) The spawning season is between May to August (Poulsen and Valo- Jøgensen, 2011)

The spawning habitats consist of rapids and sandbanks interspersed with deep rocky channels and pools where exposed roots systems serve as a substrate for egg deposition (Touch 2000)

Striped catfish is also a migratory species in Mekong River basin Once spawning

is over, the adult fish migrate downstream for feeding in the floodplains in Cambodia and Vietnam, and followed by the fingerlings In September, the adult fish are the first to leave the floodplains while the young fish of the year and

other sub-adult fish tend to stay longer in the floodplains (Srun and Ngor, 2000;

Van Zalinge et al., 2002)

2.1.2 Reproduction

2.1.2.1 Hormones

The studies in recent years show the important role of the hormone involved in regulating the process of gonad development, particularly yolk formation, maturation and ovulation

Developments of gonadal ovary are usually divided into 6 phases (Sakun and Butskaya, 1978) In which stages III, IV and the transition from phase IV to V, the growth of oocytes is controlled by pituitary hormone and follicle cells are receiving the stimulation of hormones to produce steroid hormones that control corresponding create yolk and ovulation (Nguyen Tuong Anh, 1999)

Figure 2.2 Diagram of hormones stimulating maturation and ovulation of oocyte (Nguyen Tuong Anh, 2006)

 GONADOTROPHIC (Pituitary gland, HCG, PMS) FOLLICLES

GTH II- maturational

Ganadotropin

MPF OOCYTES

PITUITARY

Ovaprim

 GnRH-A (LHRH-A , sGnRH-A, Buserelin)

 Dopamine

antagonic (Doperidone, Pimozide)

The ecological factors (Rain, temperature, pheromone)

 Antiestrogen (Clomiphen, Tamoxiphen)

 Aromatase Inhibitor

(Fadrozole, Letrozole)

The Senses BRAIN Hypothalamus

2.1.2.2 Development of Oocytes

The identities of pituitary hormones that regulate ovarian recrudescence have not been established Although the pituitary content of maturational gonadotropin (GTH II) increases during ovarian recrudescence in croaker the hormone remains barely detectable in the plasma (Copeland and Thomas, 1989)

The second gonadotropin with has recently been isolated from croaker GTH I, is more likely candidate for the vitellogenic GTH (Copeland and Thomas, 1993) In addition, two other pituitary hormones purified from sciaenid growth hormone and somatolactin stimulate ovarian steroidogenesis and may have physiology roles during oocyte growth (Safford 1992, Sigh and Thomas 1991) The hepatic production of vitellogenin, the precursor of the yolk protein, and appears to be regulated primarily by oestradiol in croaker, spotted sea trout and orangemouth

corvina (Trant 1987, Copeland and Thomas 1988; Smith and Thomas et al, in

press), although the possible involvement of other hormones has not been examined Treatment of sea trout with oestradiol induces production of vitellogenin and also the hepatic oestrogen receptor, an intermediary in the oestrogen regulation of vitellogensis (Copeland and Thomas 1988; Smith and Thomas 1990) The 2 month period of oocyte growth in sea trout coincides with

seasonal increases in plasma concentrations of the oestrogen receptor (Thomas et

al, 1987; Smith and Thomas 1991)

Although plasma testosterone levies are also elevated during this period Testosterone is unlike to be directly involved in vitellogenin because the oestrogen receptor is highly specific for oestrogen and shows negligible binding

to androgens (Smith and Thomas, 1990) Plasma oestradiol levels are maximal at the end of ovarian recrudescence in all three species and decline prior to

spawning (Thomas et al, 1987; Trant 1987) However, plasma concentration of

the steroid are maintained at half maximal values and persists in the multiple spawning sea trout throughout the remainder of its 6 month spawning season

(Thomas et al, 1987; Smith and Thomas 1991) Oestradiol concentrations also

show marked daily fluctuation in this species with peak values in the early

morning (Thomas et al, 1987)

2.1.2.3 Final oocyte maturation and ovulation

Final oocyte maturation, ovulation and spawning are initiated by a surge in GTH

II secretion from the pituitary Plasma GTH II levels increase several fold in croaker within 1 h of injection with an ovulatory dose of des-Gly[D-Ala6]-luteinizing hormone releasing hormone ethylamide and remain elevated for at least another 24h at with time the oocytes have completed maturation and are within a few hours of ovulation (Copeland and Thomas, 1989) Prolonged elevations of GTH II have also been observed in sea trout, red drum and orangemouth corvina following LHRHa or salmon ganadotropin releasing hormone analogue (sGnRHa) injection (Copeland and Thomas, 1992) GTH II levels were elevated on the fifth day of spawning in corvina but had declined to

undetectable levels 9 day later after spawning had ceased (Thomas et al,

unpublished data) The persistent elevation of GTH II in sea trout and Corvina after injection with LHRHa may account for the spawning frequency observed

GTH II performs two different functions during two temporally distinct stages of final oocyte maturation in croaker and sea trout GTH II initially induces priming

of the follicle-enclosed oocytes so that they become competent to undergo final maturation in response to the maturation-inducing steroid (MIS) (Pantino & Thomas 1990; Thomas & pantino 1991) during the GVBD (Germinal Vesicle Breakdown) phase; GTH II stimulates MIS synthesis, the traditional role ascribed

to GTH II during final oocyte maturation (Trant & Thomas, 1989; Pantino & Thomas, 1990) One of critical events during GTH II priming is induction of the ovarian membrane receptor for the MIS (Pantino & Thomas 1990; Thomas & Pantino 1991)

2.2 Maturation inducing steroid 17,20P 3-ones)

(17α,20β-dihydroxy-4-pregne-Extensive evidence indicates that 17α, 20β, 21-trihydroxy-4-pregnen-3- one (20β-S), a MIS first identified in sciaenid fishes (Trait et al, 1986), is the major MIS in croaker and sea trout and that 17α, 20β-dihydroxy-4-pregnen-3- one (17,20P) is of

minor important 20β-S is a potent inducer of final oocyte maturation in vitro; its

production coincides with the onset of the GVBD (Germinal Vesicle Breakdown) phase of final oocyte maturation, and it is he only MIS produced in large

quantities by the ovary in vitro (Trait and Thomas, 1988, 1989; Pantino and

Thomas, 1990) Moreover, the ovarian membrane receptor for 20β-S is highly specific and show negligible binding to 17,20P (Panino and Thomas, 1990)

Plasma levels of immunoreacive 20β-S rise dramatically during the later stages of final oocyte maturation in sea trout and reach peak value at ovulation whereas

17,20Plevels remain low (Thomas et al, 1987) Plasma levels of 17,20P rise in

parallel with those of 20β-S in red drum and corvina during final maturation so 17,20P could conceivably have an important function during the periovulatory period in some sciaenid species (Thomas, 1988)

17,20P (17α, 20β-dihydroxy-4-pregne-3-ones) were found in several species of the time of maturation and reproduce 17, 20 P (17α, 20β-dihydroxy-4-pregne-3-ones) have outstanding advantages are highly reactive and particularly effective

at low levels (130C) However, many studies showing 17, 20 P affect to germinal vesicle final maturation breakdown Therefore, in experiments have to use a small amount of pituitary gland or HCG preparations to ensure the sensitivity of oocytes and ovulation The female are injected 2 times: pituitary gland is injected

in the priming and decisive is injected by 17,20P There is experimental proof may be only a single injection of both 17, 20 P (17α, 20β-dihydroxy-4-pregne-3-ones) and a small amount of hormones (or pituitary gland or HCG), (Nguyen Tuong Anh, 2002)

In the world, there were many studies show that 17,20P in decisive dose has

effects on artificial propagation of some species like common carp (Cyprinus carpio) (Jalabert et al., 1977), Salmo gairdneri (Jalabert et al., 1978), Carassius auratus (Nagahama et al., 1983), Clarias batrachus (Haider & Rao., 1994), Anguilla spp (Ohata, 1996; Pederson, 2003)

In Vietnam, 17,20P is applied successfully with some carp species such as grass carp (ovulation rate: 80%, hatchability: 70%), silver barb (ovulation rate 90%, hatchability 70-80%) (Nguyen Duong Dung and Nguyen Tuong Anh, 2007) Beside, 17,20P is highly effective for catfish such as Africa catfish 86, 67-100%)

(Nguyen Tuong Anh, et al, 2000)

CHAPTER III METHODOLOGY

3.1 Time and place

- Time: The experiment began from 10-06-2012 to 01-12-2012