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)
2013
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
2013
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,
Le Hoang Phuong
i
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.
ii
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
iii
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
iv
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
v
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
vi
LIST OF ABBREVIATIONS
Ppt ...................................................................................Part per thousand
SR ...................................................................................Survival rate
LC50 ...............................................................................Lethal Concentration of 50% dead
of organism
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
vii
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
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.
2
CHAPTER 2
LITERATURE REVIEW
2.1. Biological characteristics of white leg shrimp (Litopenaeus vannamei)
2.1.1 Classification:
Phylum: Arthropoda
Class: Crustacea
Subclass: Malacostraca
Superorder: Eucarida
Order: Decapoda
Suborder: Natantia
Family: Penaeidae
Subfamily: Penaeinae
Genus: Litopenaeus
Species: Litopenaeus vannamei Boone, 1931
Figure 1: White leg shrimp (Litopenaeus vannamei)
(Source: www.fao.org)
3
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,
4
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
traditionally supplied with small frozen or processed headless shrimp from Latin
5
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
6
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.
2.3 Probiotic
Probiotics in aquaculture may act in a manner similar to that observed for terrestrial
animals.
However, the relationship of aquatic organisms with the farming environment is
much more complex than the one involving terrestrial animals.
Because of this intimate relationship between animal and farming environment, the
traditional definition of probiotics is insufficient for aquaculture.
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
benefit to the animal, but also their effect on the farming environment.
7
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.
8
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).
9
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.
10
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
Type of feed
Stage
Dry algae
(%)
Lansy ZM
(%)
Frippak
(%)
TNT100
(%)
Z1
30
40
30
0
Z2
30
40
30
0
Z3
10
20
50
20
Table 3.2: Amount of feed use for zoea stage
Fresh Algae
(lilter/m3)
50 (Chaetoceros)
(2 times/day)
Type
Commercial feed
(g/m3 )
2
(6 times/day)
Z2
50 (Chaetoceros)
(3 times/day)
2
(5times/day)
0
Z3
50 (Chaetoceros)
(3 times/day)
3
(4 times/day)
1 ÷ 2g/ 107 larvae
(1 times/day)
Stage
Z1
11
Artemia
0
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