effect of probiotics suplement in water on growth and survival rate of cobia larvae (rachycentron canadum)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES EFFECT OF PROBIOTICS SUPLEMENT IN WATER ON GROWTH AND SURVIVAL RATE OF COBIA LARVAE Rachycentron canadum By DANG DIEM TUONG A

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

EFFECT OF PROBIOTICS SUPLEMENT IN WATER ON GROWTH AND

SURVIVAL RATE OF COBIA LARVAE (Rachycentron canadum)

By

DANG DIEM TUONG

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

EFFECT OF PROBIOTICS SUPLEMENT IN WATER ON GROWTH AND

SURVIVAL RATE OF COBIA LARVAE (Rachycentron canadum)

By

DANG DIEM TUONG

A thesis submitted in partial fulfillment of the requirements for

The degree of Bachelor of Aquaculture

Acknowledgements

First of all, I wish to give my honest thank to Rectorate Board of Can Tho University, lecturers and instructors of CAF and Auburn University who have facilitated for my studying during 4.5 years in Can Tho city

By my deep gratitude I would like to give my thanks to Assoc Prof Dr Tran Ngoc Hai,

Dr Le Quoc Viet and Mr Tran Nguyen Duy Khoa who instructed me enthusiastically to finish the thesis research

For other valuable help and guide, thanks are extended to all my friends in brackish water hatchery, Nguyen Thanh Trung, Huynh The Hien, Pham Nguyen Duy, Dao Khanh Au, Nguyen Thi Tuyet Nhung and students of the course 35

I wish to express my sincere gratitude to my advisor, Dr Pham Minh Duc for his constant guidance, and thanks to all my beloved classmates in Advanced Aquaculture Program class for all great encouragement and kind help during 4.5 years in CAF

Finally, I thank my family and all my friends who have supported and encouraged me to study and finish my course

Abstract

This study aimed to evaluate the effects of different dosages of probiotics

supplemented in water on growth and survival rates of cobia larvae (Rachycentron

canadum) A triplicated experiment was conducted with differrent treatments of

probiotics including the control (without probiotics); 0.5 mg/m 3 , 1 mg/m 3 , 2 mg/m 3 The experiment was conducted in 500-L tanks holding with 2500 fry per tank (5 larvae/L), and supplied aeration continuously Brackishwater of 30ppt was used for the experiment Rotifer enriched with DHA, vitamin C and probiotics was used to feed the larvae on 3 day-post-hatch (dph) to 10 dph at 10inds/ mL Beginning on the 7dph, umbrella Artemia were suplemently fed to cobia larvae at 2-

4 inds/ mL, and on the 11 dph, enriched artemia nauplii were fed to larvae to the end

of the experiment In addition, Chlorella algae were also supplied into the nursing tank system every three days Feeding frequency was mantained three times/day at 6:00am, 12:00am, and 6:00pm Probiotics were applied to the experimental water every 3 days After 3 weeks of rearing, larvae from different treatments reach to 13.86 mm to 15.11 mm in body length There was no significant difference in length

of cobia larvae among control and treatments (p>0.05) Survival rate of fry after 3 weeks of rearing was highest (5.61 %) in treatment with 1 mg/m 3 of probiotic and lowest (3.81%) in the control which is signigicantly different from another In conclusion, probiotics help improve water quality, survival rates; reduced size variation of larvae, and relatively improved the growth of larvae It is reccommeded from this study is to apply probiotics 1g/m 3 every 3 days Further studies on the use

of probiotics and needed

Table of Contents

Acknowledgements i

Abstract .ii

Table of contents iii

List of Tables v

List of Figures v i List of Abbreviations v ii CHAPTER I INTRODUCTION 10 1.1 Background of the study 10

1.2 Objectives of the study 11 1.3 Content of the study 11 CHAPTER II LITERATURE REVIEW 12

2.1 Biological features of cobia 12 2.1.1 Classification and taxonomy 12

2.1.2 Habitat and distribution 12

2.1.3 Morphology of cobia 12

2.1.4 Food and nutrition 13

2.1.5 Reproductivity 13

2.2 Overview about the status of cobia production and study in the world 14 2.2.1 Study background of cobia Error! Bookmark not defined 2.3 Probiotics using status and study 14 CHAPTER III METHODOLOGY 16 3.1 Time and location for research 16

3.2 Study materials 16

3.2.1 Equipments for study 16

3.2.2 Water source 16

3.2.3 Feed 16

3.3 Research methodology 19 3.3.1 Experiment systems 19

3.3.2 Experimental design 19

3.3.3 Feeding methods 19

3.3.4 Management and data collection 19

3.4 Statistical analysis 21

CHAPTER IV RESULTS AND DISCUSSIONS 22

List of Tables

Table 3.1: Biochemical composition of some zooplankton 9

Table 3.2: Artemia composition 10

Table 3.3: Water quality monitoring methods 12

Table 4.1: Temperature and pH values in the cobia fry to juvenile experiment 15

Table 4.2: Growth rate of cobia larvae during 21 days 17

Table 4.3: Coefficience of variance of cobia larval length after 21 days of rearing 19

List of Figures

Figure 4.1a: Variation of temperature during the culture period 15 Figure4.1 b: Variation of TAN concentration during culture period 16 Figure 4.1.c: Variation of Nitrite concentration during culture period 16 Figure 4.2: Growth in body length of cobia larvae from different treatments during culture period 17 Figure 4.3: Distribution in body length of cobia larvae from different treatments after 21 days of culture 20 Figure 4.4: Survival rate of cobia larvae from different treatments after 21 days of culture 21

List of Abbreviations

dph Day post hatch

ppt Part Per Thousand

CFU/g Colony Former Unit

DHA decos

DLG Daily Length Gain

SGR Specific Growth Rate

SR Survival Rate

LC50 Lethal Concentration of 50% dead of organisms

CV Coefficience of Variance

HUFA High unsaturated Fatty Acid

PUFA Polyunsaturated Fatty Acid

UV Ultraviolet

Initial BLi……… Initial Body Length

Final BLf……… Final Body Length

CHAPTER I INTRODUCTION 1.1 Background of the study

In recent years, marine culture is developing in many countries as well as in Viet Nam However, technologies of marine aquaculture species production still maintain very new

activities Among many marine species, cobia (Rachycentron canadum) is considered a

potential species for marine aquaculture According to Briggs (1960), Shaffer and Nakamura (1989), cobia distributes world-wide and occur in many areas of tropical and

subtropical seas exclude the Eastern Pacific Like other marine species such as sea bass, salmon, grouper, cobia (Rachycentron canadum) is a popular species and being cultured

in many countries in the world Similar to salmon culture industry, the cobia has a potential to become a commercially high value marine culture species Moreover, the cobia has high tolerance to unfavorable conditions and potentials to be cultured in cages

in the open sea

With the advantages of a long coastline (nearly 3260km), large exclusive economic zone (more than 1 million km2) and very large area of brackish water surface area, Viet Nam has a great potiential for brackish water and marine aquaculture

In recent years, marine aquaculture in Vietnam has been developing very fast and a large number of high quality juveniles is required In order to satisfy this demand, many

research on seed production of sea bass (Lates calcarifer), grouper (Epinephelus spp) and Cobia (Rachycentron canadum) have been conducted to develop technology for hatchery

production

In the Mekong Delta, cage culture of Cobia has been developed in Kien Giang Province However, it still relies on wild seed collected from the sea In order to contribute to development of technology for Cobia seed production, this study on “Effect of probiotics

supplement in water on survival and growth rate of cobia (Rachycentron canadum)

larvae” was carried out

1.2 Objectives of the study

The specific objectives are to evaluate the effects of probiotics to the growth and survival rates of cobia larvae in order to determine suitable level of probiotics

1.3 Content of the study

Study on the effect of probiotics supplemented in water on the growth and survival rate of

cobia (Rachycentron canadum) larvae

CHAPTER II LITERATURE REVIEW

2.1 Biological f eatures of cobia

2.1.1 Classification and taxonomy

Cobia (Rachycentron canadum) is classified as the followed (Linnaeus, 1766):

Species: Rachycentron canadum

2.1.2 Habitat and distribution

Cobia, Rachycentron canadum is the only species belong to the Rachycentridae family

Cobia is widely found living from the Indo-Pacific to the southern Atlantic Ocean They usually live in many different kinds of bottom such as: mud, sand, gravel, coral bottom and even mangrove forest Cobia are eurysaline within the salinity range of 22-44 ppt but can be reared at salinity as low as 5 ppt They usually live singly or in small school (Kaiser and Holt, 2005)

Cobia usually live in the upper layer of water and can be caught in the shallow coral reefs, over sea with gravel bottom or sometimes in the estuaries They hunt for preys all days and nights around the coral reefs with many mollusk shells and sandy bottom (Do Van Khuong, 2011)

2.1.3 Morphology of cobia

Rachycentron canadum has a big size, elongated body, flatten head, mouth is terminal in

position and lower jaw is longer than upper jaw, gray color of skin with two row of white color running along the body sides They have small scales close to the skin, many single small spines at the dorsal fin

2.1.4 Food and nutrition

Cobia is a carnivorous species They feed on other fish and crustacean (crabs, shrimp) and other invertebrates They grow fast and can be up to 3-5 kg for 1 year of culture Cobia may achieve the bigger size which can be 2m in length and 61 kg in weight They can live for 15year in the natural environment The females were recorded to grow faster than the males (Kaiser and Holt, 2005)

2.1.5 Reproductivity

Cobia get maturation after 2 years old (males 1-2 years and females 2-3years) with the sizes are about 60-65 cm for males and 80 cm for females They are fully mature after 4 years The mature females spawn many times per year In the spawning season, they gather in small groups and start spawning in sea shore or open sea The fecundity of female in peak of spawning season was estimated from the range 377.000- 1.980.000 (Burns et al., 1998) Diameter of the eggs was around 1.4 mm; fertilized eggs will hatch after 24 hours (FAO, 2009)

Spawning season was mainly from April to June, and the peak of spawning is on May GSI is around 8.1-9.2% (Do Van Khuong, 2011)

Picture 2.1.5: Cobia is cultured in tank at Brackish Water Hatchery of CAF

2.2 Overview about the status of cobia production and study in the world

Cobia study was first conducted by collecting the wild cobia eggs in the off coast of North Carolina (FAO, 1990) According to many studies, cobia is a significantly potential species for aquaculture because of fast growth and good flesh quality Then, many researches about cobia grow out and reproduction was conducted from 1980-1990 in USA In 1997, technology to increase the survival rate of cobia larvae was developed in Taiwan Follow the trend of cobia culture, many countries in the world especially Asia have started to concern and did research on juvenile production in hatchery and grow out production in cages

In Vietnam, studies on marine culture and hatchery are still limited, just begun from the 1990s In 2005, the total production of marine production only achieved the number 3500 tons with some major species such as: groupers, sea bass, cobia…Comparing to the target

38 tons, it was not a very good result In 2006, total juveniles production in hatchery just reached 2 millions (Le Xan, 2005-2007, Nguyen Huu Dung, 2008 and Chang, 2008)

2.3 Probiotics using status and study

Elie Metchnikoff’s work at the beginning of this century is regarded as the first research which conducted on probiotics (Fuller, 1992) Probiotics is described as microbes ingested with the aim of promoting good health or other definition is organisms and substances contribute to intestinal microbial balance ( Parker, 1974), and later Fuller (1989) definition is a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance Now, probiotis have been used in aquaculture and the definition has also been modified In the aquatic animal, not only the digestive tract is important but also the surrounding water Probiotics are defined as microbial cells that are administered in such a way as to enter the gastrointestinal tract and to be kept alive with the aim of improving health (Gatesoupe, 1999) In another definition, probiotics were defined broadly by removing the restriction to the improvement to the intestine: “a live microbial supplement which beneficially affects the host animal by improving its microbial balance”

probiotics on aquatic animal was observed by Douillet (1993) and Douillet and Langdon

(1994) in enhancing survival and growth of Crassostrea gigas larvae The effect of

probiotics containing lactic acid bacteria in the feed was conducted by Gildberg et al (1997) on the subject was Atlantic cod fry Significant effect of Aquazyn TM-1

commercial probiotics on the water quality and growth of Cyprinus carpio was conducted

by Sharma and Bhukhar (2000) For crustaceans, study on probiotics effect on growth of

P monodon was investigated by Otta et al., (1993) The digestive tract of fish contains a

much higher number of microorganisms than the surrounding water, as many as 108 cell

g-1 (Ringo et al., 1995) As a study of Hansen and Olafsen (1998) most important being

are Pseudomonas, Cytophaga and Flexibacter which were colonized in the

gastrointestinal tract of larvae

Probiotics administration to fin fish growth and development as well as survival rate positively is well documented (Gatesoupe 2008, Wang, et al., 2008) Generally, probiotics administration during early developmental stages is the most effective (Gatessoupe, 2008) Probiotics is also well known to positively impact fish welfare (Kesarcodi- Watson

et al., 2008) by reducing the general stress response and promoting growth, as well as increasing survival in overall

In cobia seed production, there are many studies, among of these; studies related to

probiotics have been also conducted Effects of dietary chitosan and Bacillus subtilis on

the growth performance, non-specific immunity and disease resistance of cobia

(Rachycentron canadum) were investigated (Xu Geng, Xiao- Hui Dong et al., 2011) The

study has shown that specific growth rate in the fish fed on dietary treatment was significantly higher than that of the control fish (Xu Geng, Xiao- Hui Dong et al., 2011)

In Vietnam, marine species have just been studied since 1990 and is evaluated quite new (Le Xan, 2005, 2007; Nguyen Huu Dung, 2008 and Chang, 2008) For cobia seed production, many studies have just been carried out since 1998 Therefore, not so many studies about probiotics are researched However, process of cobia seed production is step

by step improved to supply seed to farmers and avoid importing seed from the foreign countries

CHAPTER III METHODOLOGY 3.1 Time and location for research

- Time: from June to December, 2012

- Location: Brackish water hatchery, College of Aquaculture and Fisheries, Can Tho University

3.2 Study materials

3.2.1 Equipments for study

- Blowers, pumps, lights, microscope, petri dishes, spoons, notes, coverage, rackets,

scale, valves, pipes, air stones

- Thermometer Salinmeter, pH, NH3/NH4+, NO2/NO3+ and alkalinity test kits

- 0.5 m3 plastic tanks x 12 used for setting the experiments,

- Chemicals: Na2S2O3, EDTA, KmnO4, Chlorine, Formalin

- Squid oil and vitamin C

3.2.2 Water source

- Freshwater source was from the municipal tap water

- Brine water (80-120ppt) were bought from Vinh Chau district, Soc Trang province, then transported and stored in brackish water hatchery in College of Aquaculture and Fisheries, Can Tho University Chlorine was used (30 - 40 ppm) to disinfect the brine water then aeration was applied continuously for at least 24 hours, after that chlorine test kit was used to check excess chlorine concentration and thiosulfate sodium was applied to neutralize before pumping through filter bag for using

- Brackish water was mixed from the freshwater and the brine water to achieve the expected salinity (30‰)

3.2.3 Feed

- Enriched rotifer (Brachionus plicatilis), Artermia (Vinh Chau and Thai Artermia)

and C hlorella algae were used to feed the larvae Biochemical composition of rotifer and Artemia is documented as in Table 3.1 and 3.2

Picture 3.1: Rotifer ( Brachionus plicatilis) using in culture

Table 3.1: Biochemical composition of some zooplankton

Table 3.2: Artemia composition

- Using vitamin C products of Bayer Company

- Probiotics (EM TAB probiotic) was used Its composition contains bacteria and the total

bacteria count is (Bacillus subtilis, Nitrobacter sp, Nitrosomonas sp) over 1.5*109 CFU/g

Picture 3.2: EM TAB Probiotics is used in water environmental tank

Cobia larvae with innitial length of 3.00 -3.30 mm were stocked at density of 10 inds/L (2500 fry/tanks) After transported from from Nam Du Island to Brackish water hatchery, College of Aquaculture and Fisheries, Can Tho University, larvae were acclimated and screened carefully before stocking

3.3.3 Feeding methods

Rotifer enriched with DHA, vitamin C and probiotics, and Chlorella algae were used to feed the larvae on 3 day-post-hatch (dph) to 10 dph at 10 inds/ mL Beginning on the 7dph, umbrella Artemia were suplemently fed to cobia larvae at 2-4 inds/ mL, and on the 11dph, enriched artemia nauplii were fed to larvae to the end of the experiment Feeding frequency was maintained three times / day at 6:00am, 12:00am, and 6:00pm

Probiotics was applied to rearing water every three days at different dosages according to the treatments Probiotic tablet was to grinded by a blender then soaked and aerated in the water for 30 minutes before applying to the rearing tanks

3.3.4 Management and data collection

During larval rearing, larval activities were observed everyday, and water was exchanged 30% every 10 days

Table 3.3.4: Water quality monitoring methods

Temperature 2 times/ day (7a.m and 5p.m) Thermometer

Nitrite Once a week (7a.m and 5p.m) Test kit

Nitrate Once a week (7a.m and 5p.m) Test kit

Salinity Once a week (7a.m and 5p.m) Salinmeter

Growth and survival rate of larvae:

Larvae samples from all rearing tanks were taken once a week (30 larvae / tank) to measure body length and to define daily length gain (DLG) and specific growth rate in TL (SGR) as the following formula

DLG (mm/day) = (L nitial – L inal )/t

SGR = (Ln L final – Ln L initial )/t x100%

For survival rates (SR), the survival rate was determined at the end of the rearing period

as the foloowing formula:

SR = (N t ÷N 0 ) * 100 %

Where L is body length, N is number of larvae, and t is culture duration (days)

3.4 Statistical analysis

Data collected was analized for mean value, standard deviation with excel software, and using one -way ANOVA (DUNCAN) method with SPSS 16.0 for windows