the effects of probiotics on quality postlavae of white leg shrimp (litopenaeus vannamei)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES Le Hoang Phuong A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Aquaculture THE EF

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

Le Hoang Phuong

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture

THE EFFECTS OF PROBIOTICS ON QUALITY POSTLAVAE OF WHITE LEG SHRIMP

(Litopenaeus vannamei)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

Le Hoang Phuong

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture

THE EFFECTS OF PROBIOTICS ON QUALITY POSTLAVAE OF WHITE LEG SHRIMP

(Litopenaeus vannamei)

Supervisor

Dr PHAM MINH DUC

Dr CHAU TAI TAO

ACKNOWLEDGEMENTS

First of all, I want to give my honest thank to Rectorate Board of Can Tho University, lectures and instructors of Course of Aquaculture and Fisheries and Auburn University who have facilitated during my studying process in Can Tho city

Secondly, I also want to give my deep gratitude to my supervisor, Dr Pham Minh Duc and Dr Chau Tai Tao for valuable guidance, advice, and encouragement Finally, I would like to give many thank to all my friends in crustacean hatchery, my classmate in advanced aquaculture class that help and encourage me when I do thesis

The author,

ABSTRACT

This study aim to evaluate the effects of 3 different types of commercial probiotics (Zimovac, DeoCare, Ecomarine) supplemented in water on growth, survival rate, and quality of white leg shrimp larvae A triplicated experiment was conducted with different treatments of probiotics including the control (without probiotics) The experiment was conducted in 100-L tanks holding 15,000 larvae (150 larvae/L), and supplied aeration continuously Brackish water of 30 ppt was used for the experiment Beginning at Nauplius3 (N3) stage, after larvae transformed to Zoea1 (Z1), the probiotics was added to treatment and re-added every 3 days During rearing process, the survival rate and length were evaluated at Z3, M3 and PL12 of development stage, beside that time of metamorphosis was investigated when larvae transform to M1 and PL1 of development stage At the end of experiment, formalin shock and salinity shock was conducted to test the quality of PL12 At zoe3 stage, there was no significant different of survival rate among control treatment and treatments used probiotics, but there was significant different (p<0.05) of length among 2 groups of treatments above, the length of larvae in control treatment had higher value than treatments used probiotics At M3 and PL12, there was significant different of survival rate and length among control treatment with treatments used probiotics, at PL12 the highest value of survival rate is 47.49% in treatment used Zimovac and lowest is 17.58% in control treatment, length of PL12 reached to 8.21mm at control treatment is higher value than 7.83 mm in treatment used Zimovac Overall, treatments use Zimovac and DeoCare had better water quality, survival rate, and the larvae had higher tolerance with stress test than others treatment It

is recommended from this study to apply Zimovac and DeoCare with dose and frequency base on production for rearing white leg shrimp

CONTENTS

CHAPTER 1 INTRODUCTION 1

1.1 Introduction 1

1.2 Research Onjectives 2

1.3 Research content 2

CHAPTER 2 LITERATURE REVIEW 3

2.1 Biological characteristics of whiteleg shrimp(Litopenaeus vannamei) 3

Classification 3

Distribution 4

Life Cycle 4

Feeding Habits of white leg shrimp 4

2.2 Studies on rearing larvae of whiteleg shrimp 5

2.3 Whiteleg shrimp production 8

2.4 Probiotic 9

CHAPTER 3 MATERIALS AND METHODS 10

3.1 Time and location 10

3.2 Materials and equipment 10

3.3 Experiment design 10

3.4 Feeding schedule 11

3.5 Collecting data 13

3.6 The method for data analysis 14

CHAPTER 4 RESULTS AND DISCUSSIONS 16

4.1 Water quality parameters 17

4.2 White leg shrimp parameters 19

4.3 Stress Tolerance 21

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 23

5.1 Conclusions 23

5.2 Recommendations 23

References 24

LIST OF TABLES

Table 3.1: Feed ingredients for Zoe stage 13

Table 3.2: Diets for Zoe stage 13

Table 3.3: Feed ingredients for Mysis stage 13

Table 3.4: Diets for Mysis stage 13

Table 3.5: Feed ingredients for Postlavae stage 14

Table 3.6: Diets for Postlavae stage 15

Table 4.1: Alkalinity value during experiment period 17

Table 4.2: Total Ammonia Nitrogen value during experiment period 18

Table 4.3: Nitrite value during experiment period 19

Table 4.4: Time of Metamorphosis of larvae at different stage 20

Table 4.5: Survival of larvae after exposed with stress test 22

LISTS OF FIGURES

Figure 1: White leg shrimp (Litopenaeus vannamei) 5

Figure 2: Variation of Temperature during rearing period 16

Figure 3: Variation of pH during rearing period 17

Figure 4: Length of larvae at different development stage 20

Figure 5: Survival rate (%) of larvae at different development stage 21

Z1 Zoea1 stage of larvae

M1 Mysis1 stage of larvae

PL1-12 Postlarvae1-12 stage of larvae

kH Alkalinity

NO2 Nitrate

TAN Total Ammonia Nitrogen

CHAPTER 1 INTRODUCTION

1.1 Introduction

White leg shrimp (Litopenaeus vannamei) are native species from the eastern coast

of the Pacific Latin America, this species has wide range of salinty, wide range of temperature, fast growth, great disease resistant In 1976, white leg shrimp farming began in South and Central America, then up for intensive development and reproductive success in the early 1980s Also during this time, production of white leg shrimp is intensive farming in South and Central America tend to rise but unstable epidemics occur Output reach to 193,000 tonnes in 1998, more than

143,000 tonnes in 2000 and 270,000 tonnes in 2004 (Briggs et al., 2004) With that

success, the intensive culture of white leg shrimp were introduced to Asia in the early '80s such as China (1988) and '90s such as Taiwan (1995), Philippines (1999), Thailand (1998), Vietnam (2000), Indonesia, Malaysia, India, and Cambodia (2002)

(Briggs et al., 2004)

Production of white leg shrimp (Litopenaeus vannamei), is a very important

economic activity in the overall farming system of Vietnam The practice of white leg shrimp culture is gaining popularity in most areas of Vietnam Within the overall agro-fishery-based economy of the country, the contribution of white leg shrimp production has been considered promising for creating jobs, earning foreign

exchange and supporting protein (Neilanda et al., 2001) However, there are some

impediments in shrimp culture that are about the shrimp seed A lot of study about probiotics are researched for increasing quality of shrimp seed, but in the market, there are many commercial production of probiotic for the famer can choose to produce shrimp seed with most effective economic in practical, therefore research on

seed production such as “ The effects of probiotics on quality of Postlavae of white leg shrimp “ is really necessary

1.2 Research Objectives

To find out the appropriate probiotics supply to improve production efficiency and

quality of Postlavae of white leg shrimp (Litopenaeus vannamei)

1.3 Research content

The effects of using difference probiotics on growth rate, survival rate, and quality

of Postlavae

CHAPTER 2 LITERATURE REVIEW

2.1 Biological characteristics of white leg shrimp (Litopenaeus vannamei)

Species: Litopenaeus vannamei Boone, 1931

Figure 1: White leg shrimp (Litopenaeus vannamei)

(Source: www.fao.org)

2.1.2 Distribution

The white leg shrimp is native to the Eastern Pacific coast from Sonora, Mexico in the North, through Central and South America as far South as Tumbes in Peru, in areas where water temperatures are normally >20°C throughout the year (FAO, 2003)

2.1.3 Life cycle

Adult Litopenaeus vannamei spawn in the ocean, releasing their eggs into the water

The eggs hatch into a non-feeding nauplius larva, which lasts about two days, before molting into a zoea stage (4-5 days), a mysis stage (3-4 days) and a postlavae (10-15 days), (Barnes 1983; FAO, 2011–stage durations are given for unspecified aquaculture conditions) Postlavae and juveniles tend to migrate into estuaries, while adults return to the sea for spawning (FAO, 2003)

2.1.4 Feeding Habits of white leg shrimp

According to Nguyen Trong Nho et al 2003, feeding habit of white leg shrimp

change with the developmental stages:

 Nauplius: Nutrient for shrimp absolutely from the yolk sac, untill the end of the N6

digestive peristaltic motion, preparing for phase using other nutrient resource

 Zoea: Larvae tend to filter food, continuous feeding, mainly food is phytoplankton

such as diatoms: Skeletonema costatum, Chaetoceros sp, Cossinodiscus, Nitzschia, Rhizosolenia, …

 Mysis: Larvae active prey, mainly food is zooplankton such as rotifers, copepods

larvae-N, N-brine shrimp, mollusk larvae, etc However, in fact Mysis can eat Silic

algea

 Post larvae: Shrimp active prey, mainly food such as Artemia, zooplankton,

copepods, crustacean larvae, larval molluscs, etc It should be noted that, at this

stage, they like to eat live bait The lack of food can lead to cannibalism

 From Post larvae to adult shrimp: From early mating period, shrimp expressing

omnivorous diet (tend to animals) Feed are other animals such as crustaceans,

molluscs, polychaete worms, small fish In artificial breeding,white leg shrimp larvae are fed with artificial foods and homemade foods such as egg yolks, soy milk, shrimp meat, eggs,

2.1.5 Studies on rearing larvae of white leg shrimp

The common environmental factors are most interested in that larval rearing are temperature and salinity Based on the research results of Dao Van Tri, Nguyen Thanh Vu (2005) show that the temperature is 28 – 300C, and 29-30‰ of salinity are most appropriate for larvae of white leg shrimp Besides that, according to FAO (2003), Overstocking can result in stress and in later stages, and may lead to cannibalism and reductions in water quality, especially when survival rates are high

In general, stocking rates for nauplii should be in the range of 100–250 nauplii/liter (100,000 – 250,000 per mt) of water Lower stocking densities are typically used where larvae are grown to harvest size in a single tank, while higher densities can be used where a two-tank system is used In the latter system, the larvae are typically cultured in a conical or “V” or “U”-bottomed tank at high density until PL4–5 and then transferred to flat-bottomed tanks for the later, benthic stages at reduced densities of up to 100 PL/liter

2.2 White leg shrimp Litopenaeus vannamei production

2.2.1 In the world

FAO, statistics of 2005 showed that the total farmed production of L vannamei

increased steadily from 8,000 tonnes in 1980 to 194,000 tonnes in 1998 After a small decline in 1999 and a more significant decline in 2000 due to the arrival of WSSV in Latin America, FAO data show a rapid increase in production to over 1,386,000 tonnes in 2004, due to the recent rapid spread of this species to Asia Main producer countries in 2004 such as: China (700,000 tonnes), Thailand (400,000 tonnes), Indonesia (300,000 tonnes) and Vietnam (50,000 tonnes)

The major market for shrimp is the United States of America, which was expected to import approximately 477,000 tonnes worth USD 3.1 billion in 2005, 1.8 times more than the 264,000 tonnes imported in 2000 The United States of America was

America More recently, the United States of America has looked to Asia to supply its increasing demand (1.9 kg/capita in 2004) Major suppliers to the United States of America in 2005 were Thailand, Ecuador, India, China, and VietNam However, the

rapidly increasing production of L.vannamei has led to serious price depression in

the international markets Similarly, farm gate value for 15–20 g size white leg shrimp has steadily decreased from USD 5/kg in 2000 to about USD 3.0–3.5/kg in

2005

The next most important market is the European Union (importing 183,000 tonnes in the first half of 2005), which favors small (31/40 count), whole, frozen shrimp Otherwise, Japanese market mainly requires large headless (16/20 count) shrimp

2.2.2 In Vietnam

Since 2002, the Fisheries Science Research such as Nha Trang Oceanography Institute (the broodstock source from Hawaii are provided by VietLinh company), Research Center for Aquaculture III Nha Trang has begun researching about the process for breeding of white leg shrimp (the broodstock source from Asia Hawaii Ventures Phu Yen company)

In 2003, The Ministry of Fishery ignore culture white leg shrimp because an anxious

about outbreak disease to native species such as monodon, as well as impact on

biodiversity Until 2006, Ministry allowed for culture white leg shrimp in Central and North , but still ignore with South By pressure from producer, in January 2008, the Ministry has agreed to allow culturing white leg shrimp in the Mekong Delta Although white leg shrimp was cultured around 2000, but its output is still small, only 84,320 tonnes compared with 236,492 tonnes of shrimp in 2009 (MARD, 2009) Till 2010, white leg shrimp farming has spread across North, Central, and South In particular, all of its output is derived from industrial farming Compared with tiger shrimp, white leg shrimp yields only about one third of total production and yield of shrimp whit leg shrimp predominant in the central and southern Central

is the main breeding areas of white leg shrimp, accounting for 75.40% of total production of white leg shrimp and 63.30% of the total farming area Meanwhile, the South accounted for only 17.4% of the total production and 19.00% of the total area

of farming The rest is the North with 7.20% of the total production and 18.00% of the total area of farming

Currently white leg shrimp have been adopted widely in the shrimp farming areas in the country and has effectively economic However, with the widespread adoption today, the risk of environmental pollution, spread of disease causing damage to farmers is unavoidable Therefore, organizations need to plan the breeding areas and invest to research and produce highly quality seed are very urgent

In this sense, Verschuere et al.,(2000) suggest a broader definition:

“It is a microbial supplement with living microorganism with beneficial effects to the host, by modifying its microbial community associated with the host or its farming environment, ensuring better use of artificial food and its nutritional value by improving the host's response to diseases and improving the quality of the farming environment.”

The microorganisms present in the aquatic environment are in direct contact with the animals, with the gills and with the food supplied, having easy access to the digestive tract of the animal

Among the microorganisms present in the aquatic environment are potentially pathogenic microorganisms, which are opportunists, i.e., they take advantage of some animal's stress situation (high density, poor nutrition) to cause infections, worsening in zootechnical performance and even death

For this reason, the use of probiotics for aquatic organisms aims not only the direct

Bergh et al.,(1992) observed that, when starting its first feeding, the intestinal flora

of the Atlantic halibut (Hippoglossus hippoglossus) changed from a prevalence of Flavobacterium spp to Aeromonas spp./Vibrio spp showing the influence of the

external environment and food on the microbial community of this fish

Vibrio spp., Plesiomonas shigelloides, and Aeromonas spp are the main causative

agents of diseases in aquaculture, and may even cause food infections in humans The interaction between the environment and the host in an aquatic environment is complex The microorganisms present in the water influence the microbiota of the host's intestine and vice versa

Makridis et al.,(2012) demonstrated that the provision of two strains of bacteria via food directly into the farming water of the incubators of turbot larvae (Scophthalmus maximus) promoted the maintenance of the bacteria in the environment, as well as

the colonization of the digestive tract of the larvae

Changes in salinity, temperature and dissolved oxygen variations, change the conditions that are favorable to different organisms, with consequent changes in dominant species, which could lead to the loss of effectiveness of the product

Accordingly, the addition of a given probiotic in the farming water of aquatic organisms must be constant, because the conditions of environment suffer periodic changes

Thus, the variety of microorganisms present must therefore be considered in the choice of probiotic to be used in aquaculture

Intensive farming systems utilize high stocking densities, among other stressors (e.g management), which often end up resulting in low growth and feed efficiency rates, besides of weakness in the immune system, making these animals susceptible to the presence of opportunistic pathogens present in the environment

In this sense, the effect of probiotics on the immune system has led to a large number

of researches with beneficial results on the health of aquatic organisms, although it has not yet been clarified how they act

In addition, probiotics can also be used to promote the growth of aquatic organisms, whether by direct aid in the absorption of nutrients, or by their supply

Probiotics most used in aquaculture are those belonging to the genus Bacillus spp (B subtilis, B licheniformis and B circulans), Bifidobacterium spp (B bifidum, B lactis, and B thermophilum), lactic-acid bacteria (Lactobacillus spp e Carnobacterium spp.) and yeast Saccharomyces cerevisiae (Y K Lee et al., 1999)

The benefits observed in the supplementation of probiotics in aquaculture include

(Verschuere et al.,2000; S Ziaei-Nejad et al.,2004)

1 Improvement of the nutritional value of food;

2 Enzymatic contribution to digestion;

3 Inhibition of pathogens;

4 Growth promoting factors;

5 Improvement in immune response; and

6 Farming water quality

Among the most recent studies that point to the effect of the use of probiotics for

various aquatic organisms stand those for fish (Verschuere et al.,2000) shrimps (S Ziaei-Nejad et al.,2004), mollusks (Macey BM et al.,2005) and frogs (Dias DC et al.,2010)

CHAPTER 3 MATERIALS AND METHODS

3.1 Time and location

3.1.1 Location:

The experiment was conducted at the College of Aquaculture and Fisheries, Can Tho University

3.1.2 Timing:

The experiment was conducted in 21days from 23/10 to 12/11

3.2 Materials and equipments

Brine water (70-80 ppt) is treated by chlorine (30ppm), aerated at least 24h, then it is checked and neutralized by disodium thiosulfate (Na2S2O3) before pumped through filter bag Fresh water is tap water Brackish water was mixed from the fresh water and brine water to achieve the expected salinity (30ppt)

Additional tanks: water storing and treating tank

Aerator system

Chemical: chlorine, Formalin

Measuring equipments: Test kit for pH, NO2-, NH3+, refractometer, Electrical Weight balance, Thermometer

Others: bucket, hand net, substrates, pumping machine, etc

Probiotics : Zimovac product of Vemedim Corporation Ecomarine product of Virbac Corporation Deocare® A products of Bayer Corporation

Larvae of shrimp after transported from hatchery in Can Tho city to CAF of CTU was reared in 100L tanks with 150 individual/L of density and 30ppt of salinity

3.3 Experimental design

The experiment use difference probiotics

Duration: From Nauplius to PL12

There were 4 treatments are repeated 4 times, total tanks needed are 16 tanks

Treatment 1: Control (without any probiotic)

Treatment 2: Zimovac (Lactobacilus spp.; Bacillus spp.; Nitrosomonas spp.;

Nitrobacter)

Treatment 3: Ecomarine (Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis)

Treatment 4: Deocare® A (Bacillus subtilis, Bacillus licheniformis)

(Dose base on the recommendation of production, added when larvae metamorphose

to Z1 and re-added every 3 days)

3.3 Feeding schedule

Table 3.1: Feed use for Zoae stage

Stage

Type of feed Dry algae

(%)

Lansy ZM (%)

Frippak (%)

TNT100 (%)