effects of magnesiumphosphoruspotassium containing in water on molting, growth and survival rate of the white shrimp (litopenaeus vannamei) juveniles, reared in low salinity water

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES EFFECTS OF MAGNESIUM-PHOSPHORUS-POTASSIUM CONTAINING IN WATER ON MOLTING, GROWTH AND SURVIVAL RATE OF THE WHITE SHRIMP Litopenaeu

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

EFFECTS OF MAGNESIUM-PHOSPHORUS-POTASSIUM CONTAINING IN WATER ON MOLTING, GROWTH AND

SURVIVAL RATE OF THE WHITE SHRIMP (Litopenaeus

vannamei) JUVENILES, REARED IN LOW SALINITY WATER

By TRAN THI BE GAM

A thesis submitted in partial fulfillment of the requirements for the degree

of Bachelor of Aquaculture

Can Tho, 12/2013

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

EFFECTS OF MAGNESIUM-PHOSPHORUS-POTASSIUM CONTAINING IN WATER ON MOLTING, GROWTH AND

SURVIVAL RATE OF THE WHITE SHRIMP (Litopenaeus

vannamei) JUVENILES, REARED IN LOW SALINITY WATER

By TRAN THI BE GAM

A thesis submitted in partial fulfillment of the requirements for the degree

of Bachelor of Aquaculture

Supervisor MSc HUYNH TRUONG GIANG

ACKNOWLEDGEMENT

Foremost, I would like to express my sincere gratitude to my supervisor, MSc Huynh Truong Giang for the continuous support of my study, for his invaluable guidance, advice, encouragement, and immense knowledge His guidance helped me in all the time of research and writing of this thesis I want to dedicate my great appreciation to

Ms Phan Thi Cam Tu for her kind help in finishing the research

Many thanks are also giving to all other doctors of the college of aquaculture and fisheries, and especially to those of the Department of Applied Hydrobiology for providing me with great working and learning conditions

I would love to express my sincere appreciation to many of my friends, especially Pham Thi Trang Nhung, Le Phuoc Dai, Bui Doan Luan, and Tran Trung Giang for their unconditionally kind help throughout the experimental period

Last but not the least, I really want to thank my family for their great life-time support which makes everything possible for me

Tran Thi Be Gam

APPROVEMENT

The thesis “Effect of Mg-P-K levels containing in water on molting, growth, and

survival rate of the white shrimp L vannamei juveniles, reared in low salinity

water” was completed base on my results

ABSTRACT

The objective of the present study was to evaluate the effects of several aqueous magnesium, potassium, and phosphorus on survival, growth, and molting interval of

L.vannamei at salinity 2‰ The study consisted of two experiments The study

consisted of two experiments For the first experiment with postlarvae 15-day, aqueous Mg-K-P were supplemented directly into water at three different levels (10-5-5; 20-10-10; 40-20-20 mg/L) and a treatment with no supplemented minerals severed as the control group In the second experiment, concentrations of minerals added in the treatments were the same as the first experiment but conducted with juvenile shrimp (4.7±0.02 g) Each treatment comprised of 14 shrimp Each shrimp was individually cultured in a small net cage Fourteen small net cages were allocated in a composite tank with a capacity of 1 m3 Molting interval, survival, growth rate were recorded

Results from the first experiment indicated no significant differences (p>0.05) in

survival and growth of shrimp among treatments Results from the second experiment reveal a significant difference in survival between 10-5-5 treatment (14.29%) and all other treatments (28-36%) The results showed highest weight gain (1.7±0.27 g), length gain (1.81±0.29 cm), SGR (1.71±0.35% day-1) and DWG (0.08±0.016 g day-1)

in 20-10-10 treatment However, no differences in molting interval were observed These results suggest supplementation of mineral are common in low salinity water

Keywords: Litopenaeus vannamei, low salinity, magnesium, phosphorus, potassium,

molting

TABLE OF CONTENTS

Pages

ACKNOWLEDGEMENT i

APPROVEMENT ii

ABSTRACT iii

TABLE OF CONTENTS iv

LIST OF TABLES vii

LIST OF FIGURES viii

LIST OF FIGURES viii

LIST OF ABBREVIATIONS ix

LIST OF ABBREVIATIONS ix

Chapter 1 1

INTRODUCTION 1

1.1 Problem identification: 1

1.2 Objective: 2

1.3 Research content: 2

Chapter 2 3

LITERATURE REVIEW 3

2.1 Biological characteristics of white shrimp (Litopenaeus vannamei): 3

2.1.1 Classification: 3

2.1.2 Distribution: 3

2.1.3 Life cycle: 3

2.1.4 Growth characteristics: 4

2.1.5 Molting cycle: 4

2.1.6 Nutrition demand: 6

2.2 Osmoregulation of shrimp 7

2.3 Effect of low salinity on the growth and survival rates of white shrimp (L.vannamei) 8

2.4 White shrimp (L vannamei) production in the world: 8

2.5 White shrimp (L vannamei) production in Vietnam: 9

Chapter 3 10

MATERIALS AND METHODS 10

3.1 Time and location 10

3.2 Materials 10

3.2.1 Shrimp culture 10

3.2.2 Equipment and chemicals 10

3.3 Research design and experimental approaches 10

3.3.1 Experiment 1: Effects of Mg-P-K supplemented in water on growth and survival rate of white shrimp (L vannamei) 10

3.3.2 Experiment 2: Effects of Mg-P-K supplemented on molting intervals of white shrimp L vannamei (4-5 g) that reared in low salinity water 12

3.4 Data analysis 13

Chapter 4 14

RESULTS AND DISCUSSIONS 14

4.1 Experiment 1: Effects of Mg-P-K supplemented in water on growth and survival rate of white shrimp (L vannamei) 14

4.1.1 Water quality 14

4.1.1.1 Temperature (oC) and pH 14

4.1.1.2 Total ammonia nitrogen (TAN), Nitrite (N-NO2-), and Phosphate (P-PO43-) 14 4.1.1.3 Calcium (Ca2+), Magnesium (Mg2+), Potassium (K+) concentrations and Mg:Ca

4.1.2 Growth performance 16

4.1.2.1 Weight Gain (WG), Daily Weight Gain (DWG), Specific Growth Rate (SGR) 16

4.1.2.2 Length Gain (LG), Daily Length Gain (DLG), Specific Growth Rate (SGR) 17

4.1.3 Survival rate (%) 17

4.2 Experiment 2: Effects of Mg-P-K supplemented on molting intervals of white shrimp L vannamei that cultured in low salinity water 19

4.2.1 Water quality 19

4.2.1.1 Temperature, pH 19

4.2.1.2 Total ammonia nitrogen (TAN), Nitrite (N-NO2-), Phosphate (P-PO43-) .19

4.2.1.3 Calcium (Ca2+), Magnesium (Mg2+), Potassium (K+), and Mg:Ca ratios .20

4.2.2 Growth performance of white shrimp L vannamei 21

4.2.2.1 Molting intervals of white shrimp L vannamei 21

4.2.2.2 Weight gain of white shrimp after molting 22

4.2.2.3 Length gain of white shrimp after molting 23

4.2.2.4 Growth performance of white shrimp at the end of experiment 24

4.2.2.5 Survival rate 25

Chapter 5 27

CONCLUSIONS AND RECOMMENDATIONS 27

5.1 Conclusions 27

5.2 Recommendations 27

REFERENCES 28

LIST OF TABLES

Table 1: Events of possible technological significance occurring in the molting cycle

of shrimp 5

Table 2: Ionic composition of seawater and freshwater 7

Table 3: Production of white shrimp in North, Central and South of Viet Nam in 20099 Table 4: Methods for water quality sampling and analysis 11

Table 5: Temperature and pH in Experiment 1 14

Table 6: TAN, N-NO2- and P-PO43- in Experiment 1 15

Table 7: K+, Mg2+ and Ca2+ concentrations in Experiment 1 15

Table 8: Hardness and Mg:Ca ratios for white shrimp reared in low salinity water in Experiment 1 16

Table 9: Growth performance indices of white shrimp L.vannamei that reared in salinity 2‰ with different Mg-K-P supplemented levels in Experiement 1 .17

Table 10: Initial length (cm), final length (cm), daily length gain (cm day-1), specific growth rate (% day-1), and length gain (g) for L.vannamei reared in salinity 2‰ in Experiment 1 17

Table 11: Temperature and pH in Experiment 2 19

Table 12: TAN, Nitrite, and Phosphate for white shrimp reared in low salinity water in Experiment 2 20

Table 13: Potassium, magnesium, and calcium for white shrimp reared in low salinity water in Experiment 2 20

Table 14: Hardness and Mg:Ca ratios for white shrimp reared in low salinity water in Experiment 2 21

Table 15: Weight gain (g), daily weight gain (g day-1), specific growth rate (% day-1) of shrimp reared in low salinity water in Experiment 2 25

Table 16: Length gain (cm), daily length gain (cm day-1), specific growth rate (% day -1 ) of shrimp reared in low salinity water in Experiment 2 25

LIST OF FIGURES

Fig 1: White shrimp (Litopenaeus vannamei) 3

Fig 2: Life cycle of Litopenaeus vannamei 4

Fig.3 Net cage used in Experiment 2 13

Fig.4: Survival rate (%) of shrimp that reared in low salinity (2‰) at different Mg-K-P supplements Each bar represents the mean value from 3 replicates with a standard error 18

Fig.5: Molting intervals of white shrimp (L.vannamei) 22

Fig.6: Weight gain of L.vannamei after molting 22

Fig.7: Mean weight of shrimp after each molting 23

Fig.8: Length gain of L.vannamei after each molting 23

Fig.9: Mean length of shrimp after each molting 24

Fig 10: Survival rate (%) of shrimp in Experiment 2 26

FAO Food and Agriculture Organization

Chapter 1 INTRODUCTION 1.1 Problem identification:

White shrimp (Litopenaeus vannamei) had been exploited and appeared on the

consumer market of the United State since 1709 Currently, they are widely cultured in countries of Asia-Pacific The introduction of white shrimp to Asia has given rise to a bloom in farming of this species in China, Thailand, Indonesia and Viet Nam in the last decade, resulting in an almost complete shift from the native black tiger shrimp

(Penaeus monodon) to this introduced species in Southeast Asia In the year 2010,

global aquaculture for this species reached 2.7 million metric tons (MT) (FAO, 2012)

In Viet Nam, cultivation of white shrimp L vannamei culture has been growing

rapidly and become an important economic activity in Mekong Delta In fact, white shrimp production has been increasing since 2000 and the export value reached 676.6 million USD (177,817 MT) in 2012 (VASEP, 2013)

There are many problems which farmer needs to care for culturing white shrimp Requirement nutrients of white shrimp, especially minerals, are most important Around twenty-two minerals, both macro and micro, have been found essential to animals, fish and shrimp However, unlike fin-fishes, there is relatively fewer information on the mineral needs of shrimps Most of the information available for mineral requirements has been done under laboratory conditions using purified or semi-purified diets and scanty information is available on elemental requirements under practical culture conditions (Tacon, 1987)

White shrimp L vannamei has rapid growth, short molting cycle cause high demand

minerals Molting, especially, is fundamental for growth in shrimps Shrimp generally lose most of their body content of minerals during molt and replace it from the water White shrimp live in an environment that is hypertonic and continually drink small amounts of water and thus they can absorb some minerals directly from the water via the gills, skin or both (Tacon, 1987) Although minerals are very important, the study

of mineral requirements of white shrimp has been quite neglected Besides that, the

ability of white shrimp L.vannamei to tolerate a wide range of salinities (0.5-40 ‰)

has made it a popular species for low salinity culture However, in low salinity water, problems still arise from deficiencies in minerals of pond water Therefore, direct supplementation of macro-minerals as magnesium, phosphorus, and potassium in the water instead of food are of primary concern In this study, molting stage durations,

growth performance indices and survival rate of white shrimp L vannamei were

examined

1.2 Objective:

This study was undertaken on L.vannamei to evaluate the effect of Mg-P-K levels

containing in low salinity water in molting cycle, growth, and survival rate in order to

propose potential use for white shrimp L vannamei culture in low salinity area in

Mekong Delta

1.3 Research content:

- Research on effects of different Mg-P-K levels on growth and survival rate in

white shrimp L vannamei postlarvae

- Research on effects of different Mg-P-K levels on molting intervals of white

shrimp L vannamei juveniles

Chapter 2 LITERATURE REVIEW

2.1 Biological characteristics of white shrimp (Litopenaeus vannamei):

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 postlarva (10-15

days) (Barnes 1983; FAO, 2011 – stage durations are given for unspecified aquaculture conditions) Postlarvae and juveniles tend to migrate into estuaries, while adults return to the sea for spawning (FAO, 2011)

Fig 2: Life cycle of Litopenaeus vannamei (Bailey-Brock and Moss, 1992)

2.1.4 Growth characteristics:

Males become mature from 20 g and females from 28 g onwards at the age of 6–7

months L vannamei weighing 30–45g will spawn 100,000–250,000 eggs of

approximately 0.22 mm in diameter Hatching occurs about 16 hours after spawning and fertilization The first stage larvae, termed nauplii, swim intermittently and are positively phototactic Nauplii do not feed, but live on their yolk reserves The next larval stages (protozoea, mysis and early postlarvae respectively) remain planktonic for some time, eat phytoplankton and zooplankton, and are carried towards the shore

by tidal currents The postlarvae change their planktonic habit about 5 days after molting into postlarvae, move inshore and begin feeding on benthic detritus, worms, bivalves and crustaceans (FAO, 2012)

2.1.5 Molting cycle:

Molting in shrimp is a phenomenon that always occurs in the process of shrimp culture The size of shrimp meat grew while the outer shell does not grow large, so for the adjustment, the shrimp will release the old shell and reshape a new shell with conducted by calcium This molting process resulted in increased body size periodically After a hard outer shell, shrimp body size remains until the next molting cycle

Duration of the molting cycle depends on the species and age of the shrimp Molting frequency on white shrimp decreased along with increasing the size of shrimp The larval stage, the white shrimp molting every 40 hours at 28 oC while juveniles

weighing 1-5 g, molting every 4-6 days The next on the weight of 15 g, the period of molting occurs every 2 weeks (Cortell, 2012)

Molting phase is the most important phase for shrimp, because at this phase, the shrimp in a most weak condition and not yet hardened outer shell layer, so making it very susceptible to diseases infection and attacked by other shrimps (cannibalism) and predators Besides susceptible to diseases and predators, in molting phase, the shrimps are also very vulnerable to environmental changes, whether environmental changes that occur in ponds and the changes that occur because of weather changes

Shrimp have to face the pervasive influence of the molt cycle on their internal environment during their entire life cycle (Passano, 1960; Bliss, 1985; Garcia, 1988; Franco et al., 2006)

Table 1: Events of possible technological significance occurring in the molting cycle of shrimp

( Cobb and Bryant F, 1977 )

increasingly harder

- Continued water absorption

- Tissue growth begins

- Accumulation of melanin precursor (storage form of N-acetyldopamine) and other organic reserves

- Ecdysial sutures open

2.1.6 Nutrition demand:

- Protein: Protein, which is required for growth and maintenance, is an expensive

component in a diet Salinity also is among the factors known to influence the protein requirement of shrimp (Boonyaratpalin, 1996) Some studies comparing the optimum dietary protein level for shrimp For juveniles, Colvin and Brand (1977) reported less than 30% to be the protein requirement while Kureshy and Davis (2002) found a

maximum protein requirement at 32% for juveniles and sub-adults of L vannamei

The first report of protein requirement for post-larvae, raised in tanks during one month (Colvin and Brand, 1977), indicated 30-35%

- Phospholipids and cholesterol are two essential lipids for penaeid shrimp

Cholesterol is an essential lipid that is closely involved in the process of molting in shrimp (Kanazawa et al 1975) and is important in enhancing shrimp growth (Gong et

al 2000) Phospholipids may be contributive to facilitating molting process as well Cholesterol requirement can be lowered as dietary phospholipid level increases A combination of 0.05% cholesterol and 5% phospholipid is recommended for intensive

L vannamei culture (Gong et al., 2001)

- Minerals: Marine shrimps absorb minerals from their aquatic environment aside

from the minerals that come from the food they eat (F Piedad-Pascual, 1989) Minerals are essential components of bones, teeth, fin, and exoskeleton These are necessary for maintenance of osmotic pressure, acid-base balance, thus the regulation

of pH of blood, haemolymph, urine, and other body fluids They are also components

of soft tissues, enzymes, some vitamins, hormones and respiratory pigments and are essential for muscle contraction and transmission of nerve impulses (Davis, 1996) Shrimps need minerals for growth because of repeated molting wherein minerals are lost (Kanazawa, 1985)

 Phosphorus: Phosphorus is an element shrimp cannot find in reasonable

amount in seawater In contrast to calcium, phosphorus has to be added to the diet, but according to the pH of the anterior part of the stomach differences are expected in relation to the nature of the phosphate salt (Velasco et al., 2001)

As a component of these important biological substances, phosphorus plays a central role in energy and cell metabolism

 Magnesium: magnesium plays a role in the normal metabolism of lipids,

proteins, and carbohydrates serving as a cofactor in a large number of enzymatic and metabolic reactions (Davis and Lawrence, 1997) Seawater typically contains high levels of magnesium is excreted by marine crustacean and fish, resulting in blood levels lower than of the external medium (Dall and Moriarty, 1983) Cheng et al (2005) reported a dietary Mg2+ requirement for

optimal growth of 2.6-3.46 g Mg2+ kg-1 for L.vannamei reared in low salinity

waters

 Potassium: Compared with the other essential ions, potassium is a minor

constituent in brackish and freshwater (Horne, 1969), but it plays important role

in normal growth, survival, and osmoregulatory function of crustaceans (Mantel and Farmer, 1983) When reared in potassium deficient seawater, L.vannamei

displayed anorexia, low activity, poor growth and even death (Zhu et al., 2004)

 Calcium: the calcium requirement may be totally or partially met though

absorption of calcium from the water (Deshimaru and Yone, 1978) The regulatory mechanism for calcium is obviously presented with an added challenge during the molt cycle of crustaceans, when there are large fluxes of calcium Among various species, the concentration of total calcium consistently shows an increase during the period prior to the molt (Greenaway, 1985)

Table 2: Ionic composition of seawater and freshwater

Huong and Wilder (2008) reported hemolymph osmolality of the shrimp (Litopenaeus vannamei) exposed to salinities of 0.5‰ or 1‰ decreasing rapidly from 800 mOsm to

540 mOsm after 6 hours Levels also dropped dramatically from 800 mOsm to 560 mOsm in shrimp exposed to 3‰ after 6 hours and 1 day Hemolymph osmolality of the white shrimps changed after exposure to low salinities, showing hyper-

osmoregulatory behavior in low salinities The white shrimp (Litopenaeus vannamei)

can not survive in low salinities (<1‰) because it loses the capacity to osmoregulate

2.3 Effect of low salinity on the growth and survival rates of white shrimp

(L.vannamei)

The ability of L.vannamei to tolerate a wide range of salinities (0.5-40 ppt) has made it

a popular species for low salinity culture (McGraw et al., 2002; Samocha et al., 1998;

2002) The high tolerance of L.vannamei to low salinity and the year-round availability

of healthy postlarvae make this species an excellent candidate for inland farming

Therefore, recently, farmers have been focusing effort on culturing L.vannamei in low

salinity environments

In lower salinity culture, there is an osmotic pressure between the shrimp body and the surrounding water, resulting in automatic water uptake mainly through gills and intestine With lower salinity the shrimp face more difficulties to uptake minerals from the water As nutrient intake directly and indirectly influences shrimp’s tolerance of stress, the supplementation of selected minerals and lipids above the levels typically used in marine shrimp feeds, beside supplementation in water directly

There is evidence from short-term exposures in culture tanks that both potassium and magnesium additions to well water will enhance survival and growth of postlarvae (Davis et al 2005; Roy et al 2007a) Roy et al (2007a) reported an increase in shrimp growth when potassium levels were raised in low salinity waters To date, several authors have established that when raising shrimp and other marine species in low salinity waters it is important to maintain sodium to potassium ratios (Na : K) at levels similar to seawater diluted to the same salinity (Fielder et al 2001; Davis et al 2004; Zhu et al 2004; Roy et al 2007a)

2.4 White shrimp (L vannamei) production in the world:

Litopenaeus vannamei was introduced into Asia experimentally from 1978-1979, but beginning in 1996, L vannamei was introduced into Asia on a commercial scale This

started in Mainland China and Taiwan Province of China and subsequently spread to the Philippines, Indonesia, Viet Nam, Thailand, Malaysia and India (RAP, 2004)

In 2008, 67% of the world production of cultured penaeid shrimp (3,399,105 tones)

consisted of L vannamei (2,259,183 tones) Such dominance was attributed to an

18-fold increase of production in Asia, from 93,648 MT in 2001 to 1,823,531 MT in

2008, which accounts for 82% of the total world production of L vannamei China leads the world cultured L vannamei production from 33% in 2001 to 47% in 2008

(1,062,765 MT), among which 51% (542,632 MT) were produced in inland freshwater pond (Liao and Chin, 2011) Thailand produced 299,000 tones, Vietnam 100,000 MT,

Indonesia 103,874 MT of L.vanamei in 2005 (Kongkeo, 2007)

2.5 White shrimp (L vannamei) production in Vietnam:

Table 3: Production of white shrimp in North, Central and South of Viet Nam in 2009

(MARD - Ministry of Agriculture and Rural Development)

In Viet Nam, white shrimp cultured from 2000 but low production, reached 84,320

MT (MARD, 2009) However, in recent year, cultivation of white shrimp L vannamei

culture has been growing rapidly and become an important economic activity In 2012, total exports of white shrimp were valued at 676.6 million USD (VASEP, 2013) In Mekong Delta, white shrimp reached 77,830 MT with 15,727 ha (VASEP, 2013)

Chapter 3 MATERIALS AND METHODS 3.1 Time and location

The research was carried out in the Department of Applied Hydrobiology, College Aquaculture and Fisheries from July 2013 to November 2013 Water sample was analyzed at the Laboratory for Water Quality Study, College Aquaculture and Fisheries, Can Tho University

3.2 Materials

3.2.1 Shrimp culture

Postlarval L.vannamei were obtained from a hatchery in Can Tho, and transport to the

laboratory of the Department of Applied Hydrobiology, College Aquaculture and Fisheries, Can Tho University Shrimp were acclimated down to low salinity water (2

‰) over a week at 27 oC During acclimation period, shrimp were fed to satiation twice daily with commercial feed diet 40% protein During the first three days,

postlarval were offered a combination of Artemia

3.2.2 Equipment and chemicals

 Chemicals: Na2S2O3, Na2[Fe(CN)5NO].2H2O, Fe(NH4)(SO4)2.6H2O, C2H5OH, NaOH, C6H5OH, EDTA, NaOCl, Patton-Reeder indicator, SnCl2, H2SO4, Bradford solution, BSA are purchased from Merck (Germany) and Sigma (St Louis, MO, USA) The other chemicals and reagents used are of analytical grade

 Equipment: aerators, 100L-tanks, bucket, hand net, substrates, pumping machine; thermometer, microscope ZX21, refactormeter, multiparameter YSI

556, spectrophotometer UV-Vis Thermo Helios (USA), quartz and glass 1 cm cuvettes, digital camera microscope, and others

3.3 Research design and experimental approaches

3.3.1 Experiment 1: Effects of Mg-P-K supplemented in water on growth and survival rate of white shrimp (L vannamei)

This experiment includes four treatments with four different levels of Mg-P-K added

- Treatment 1: without Mg-P-K supplemented (control)

aeration system Magnesium, phosphorus and potassium were also determined before shrimp were stocked Shrimp were fed their respective diets at 7:00, 14:00, and 21:00h Amount of feed based on shrimp feeding requirement through observation of feeding activity of shrimp Salinity water was 2‰ At the end of the 45-day, shrimp were harvested and survival and growth were assessed

Preparation of test solution

Mg2+ test solutions were prepared by dissolving 8.4 g MgCl2.6H2O in 20 mL of distilled water to prepare 1,000 mg/L Mg2+ stock solution Similarly, P and K concentration in the tanks were prepared by dissolving 3.87 g NaH2PO4 and 1.91 g KCl respectively to make 1,000 mg/L for each stock solution

Then, these stock solutions were diluted with seawater to make various concentrations corresponding to each treatments using the equation CV=C’V’ at salinity of 2‰

Water quality parameters were tested:

Temperature, pH, salinity, TAN, NO2-, PO43-, Ca2+, Mg2+, K+ concentrations base on the methods as described in Table 4

Table 4: Methods for water quality sampling and analysis

End of the experiment

Shrimp performance indices are measured:

i f 1

T T

W W ) day (g DWG

i f 1

T T

L L ) day (g DLG

T

LnWLnW

)day(%

SGR

i f

i f

- Survival rate, SR:

100N

NN(%)SR

i

f i

low salinity water (2 ‰) over a week in a composite tank Shrimp were fed ad libitum twice daily with a commercial shrimp diet 40% protein (Grobest Co Ltd, Vietnam) The weight of the shrimp range 4.47±0.02 g, averaging with no significant size difference among treatments Only healthy shrimp were used for the study

Each treatment comprised of fourteen shrimp Each shrimp was individually cultured

in a net cage Fourteen small net cages were allocated in the composite tank with the capacity of 1 m3 Therefore, total was four tanks in this experiment Experimental cage design described as Fig 3

Fig.3 Net cage used in Experiment 2

Water was renewed weekly Before shrimp was allocated in experimental tanks, water samples were collected for water quality test (parameters are showed in Table 4) Shrimp were fed at 7:00, 14:00 and 21:00 h Amount of feed based on shrimp feeding requirement through observation of feeding activity of shrimp Salinity water was 2‰

Determination of molting intervals of shrimp:

Molting of shrimp were recorded daily at 7~8:00 AM through the old shell in the net case Shrimp molting interval as expressed as days between 1st molting and next molting Two molting intervals were recorded

Water quality parameters were tested: temperature, pH, Mg, P, K Sampling frequency

and methods for examination for these parameters are the same as those of

Experiment 1 (Table 4)

3.4 Data analysis

A multiple-comparison test (Tukey’s) was used to examine if significant differences among treatments using the SAS computer software (SAS Institute, Cary, NC, USA) All values in percentage (survival rate) are arcsine-transformed to satisfy the requirement for the normal distribution Statistical significance of differences required

Chapter 4 RESULTS AND DISCUSSIONS 4.1 Experiment 1: Effects of Mg-P-K supplemented in water on growth and

survival rate of white shrimp (L vannamei)

4.1.1 Water quality

4.1.1.1 Temperature ( o C) and pH

Temperature ranged from 25.2-28.8 oC and no significant difference was found among treatments pH ranged from 7.5-8.6 (Table 5) Wyban et al (1995) suggest that temperature optimum is about from 25 to above 30oC Moreover, Wang et al (2004)

recommended the favorable pH range of 7.6-8.6 for L.vannamei Hence, temperature

and pH in treatments were suitable for shrimp growth

Table 5: Temperature and pH in Experiment 1

27.4±0.7 (25.7 - 28.6)

8.3±0.16 (7.6 - 8.5)

8.3±0.17 (7.8 - 8.5)

10-5-5

26.9±0.7 (25.2 - 28)

27.5±0.7 (25.7 - 28.8)

8.2±0.17 (7.7 - 8.4)

8.3±0.16 (7.8-8.5)

20-10-10

27.1±0.7 (25.3 - 28.2)

27.8±0.6 (25.7 - 28.5)

8.2±0.12 (7.7 - 8.3)

8.3±0.16 (7.8 - 8.5)

40-20-20

27.2±0.6 (25.7 - 28.6)

27.6±0.7 (25.7 - 28.7)

8.2±0.18 (7.5 - 8.4)

8.3±0.18 (7.7 - 8.6)

Values represent the Mean ± Standard deviation (Min-Max) during experiment

4.1.1.2 Total ammonia nitrogen (TAN), Nitrite (N-NO 2 - ), and Phosphate (P-PO 4 3- )

TAN concentration fluctuated largely over the study period TAN concentrations ranged from 0.06 to 0.76 mg L-1 In the treatment 40-20-20, TAN concentration reached in the 21st day then gradually declined at the end of the experiment N-NO2-ranged from 0.001 to 5.87 mg L-1 The highest average N-NO2- concentration was found in treatment 40-20-20 with a value of 1.49±2.17 mg L-1 while treatment 10-5-5 and 20-10-10 were 1.12±1.16 and 1.12±1.15 mg L-1 A suitable range of nitrite for marine shrimp culture is below 1.0 mgL-1 (Boyd, 1998) Thus, NO2- concentrations in the present experiment did not cause any shrimp mortality TAN and N-NO2- concentrations are shown in Table 6

In this experiment, NaH2PO4 was added to four treatments at levels 0, 5, 10, and 20

mg L-1 phosphorus Therefore, fluctuation of phosphate reading was between 0.17 and 15.67 mg L-1 The highest average P-PO43- concentration was found in treatment 40-20-20 with a value of 14.31±1.17 mgL-1 while treatment 10-5-5 and 20-10-10 were 5.02±1.6 and 7.77±1.09 mgL-1 P-PO43- concentrations in the present experiment did not cause any shrimp mortality P-PO43- concentrations are shown in Table 6

Table 6: TAN, N-NO 2 - and P-PO 4 3- in Experiment 1

Values represent the Mean ± Standard deviation (Min-Max) during experiment

4.1.1.3 Calcium (Ca 2+ ), Magnesium (Mg 2+ ), Potassium (K + ) concentrations and Mg:Ca ratios

In this study, calcium was not supplemented into water Therefore, calcium (Ca2+) concentrations were ranged from 15.53±1.33 to 23±0.2 mg L-1 during the experiment period Magnesium (Mg2+) was added to provide concentrations of 0, 10, 20, and 40

mg L-1 per tank to three replicate tanks per treatment, respectively Consequently, the average concentrations of magnesium were 69.0±4.5, 98.5±4.6, 100.7±9.1, 120.1±10.0

mg L-1, respectively Increases in magnesium concentrations lead to increase hardness concentrations in the water The minimum hardness was recorded 275±10.5 mgCaCO3

L-1 and maximum hardness was recorded 349.8±23.0 mgCaCO3 L-1 in all the treatments (Table 8)

In this experiment, water was supplemented with 0, 5, 10, 20 mg L-1 potassium (K+) per treatment from potassium chloride (KCl) Potassium concentrations were recorded 29.5±2.1, 39.3±2.7, 43.3±7.3, and 52.4±5.1, severally (Table 7) In general, magnesium, phosphorus and potassium in treatments were suitable for shrimp growth

Table 7: K + , Mg 2+ and Ca 2+ concentrations in Experiment 1

Table 8: Hardness and Mg:Ca ratios for white shrimp reared in low salinity water in Experiment 1

Values represent the Mean ± Standard deviation (Min-Max) during experiment

Magnesium levels are also important for shrimp well-being, and can be maintained by regulating ratios of divalent cations in the water Roy et al (2007) suggested that ratios

of Mg:Ca should also approximate those found in natural seawater (3.1:1) to ensure

adequate survival of L.vannamei reared under low salinity conditions In addition,

Limsuwan (2009) reported that 3.4:1 Mg:Ca ratio is recommended in the face of medium to high salinity levels, and 5:1 for low salinities

In this study, magnesium was supplemented in the water Thus, ratios of Mg:Ca in water treatments were higher in natural seawater The ratios of Mg:Ca in control, 10-5-

5, 20-10-10, and 40-20-20 treatment were 3.3±0.37, 4.7±0.49, 5.1± 0.97, and 6.1±0.88, respectively

4.1.2 Growth performance

4.1.2.1 Weight Gain (WG), Daily Weight Gain (DWG), Specific Growth Rate (SGR)

Final weights ranged from 0.52 to 0.64 g, while DWG ranged from 0.012 to 0.014 g day-1 Shrimp in 40-20-20 treatment showed the highest SGR and WG, whereas the lowest one was control shrimp However, there were no significant differences in final weights, WG, DWG, and SGR of shrimp reared in low salinity waters containing various levels of Mg-K-P (p> 0,05) (Table 9)

Table 9: Growth performance indices of white shrimp L.vannamei that reared in salinity 2‰

with different Mg-K-P supplemented levels in Experiement 1

Values (Means±SE of 3 replicates) in the same row sharing a common superscript are not significantly different ( p> 0.05 )

4.1.2.2 Length Gain (LG), Daily Length Gain (DLG), Specific Growth Rate (SGR)

Shrimp reared in 40-20-20 mg L-1 Mg-K-P yielded the highest final length (4.24 cm), length gain (3.64 cm), daily length gain (0.094 cm day-1), and specific growth rate (4.34%), while control treatment showed lowest However, no significant difference in

LG, DLG, SGR and final length was observed among treatments (Table 10)

Table 10: Initial length (cm), final length (cm), daily length gain (cm day -1 ), specific growth rate (% day -1), and length gain (g) for L.vannamei reared in salinity 2‰ in Experiment 1

10-Studies of Roy et al (2007) reported that white shrimp L vannamei (1 g) reared in

salinity of 4 ‰ with five different levels of K+ (0, 5, 10, 20, and 40 mg L-1) supplemented in 6 weeks, Survival rates were 23.3%, 95.0%, 96.7%, 93.3%, 93.3%,

respectively However, white shrimp L vannamei reared in low salinity of 4 ‰ then

supplemented five different levels of Mg2+ (10, 20, 40, 80, 160 mgL-1, the survival rates were 60.2%, 90.0%, 96.5%, 93.2%, 95.0%, respectively

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4.2 Experiment 2: Effects of Mg-P-K supplemented on molting intervals of white

shrimp L vannamei that cultured in low salinity water

4.2.1 Water quality

4.2.1.1 Temperature, pH

Temperature ranged from 25.3-30.2 oC and no significant difference was found among treatments pH ranged from 7.6-8.7 (Table 11) Wyban et al (1995) suggested that temperature range from 25 to 30oC is considered to be good for marine shrimp Moreover, Wang et al (2004) recommended the optimal pH range for L.vannamei is

7.6-8.6 Therefore, temperature and pH in treatments were suitable for shrimp growth

Table 11: Temperature and pH in Experiment 2

Values represent the Mean ± Standard deviation (Min-Max) during experiment

4.2.1.2 Total ammonia nitrogen (TAN), Nitrite (N-NO 2 - ), Phosphate (P-PO 4 3- )

TAN concentration fluctuated largely over the study period TAN concentrations ranged from 0.03-1.06 mg L-1 In the treatment 40-20-20, TAN concentration reached

in the 7th day then gradually declined at the end of the experiment N-NO2- ranged from 0.02 to 1.43 mg L-1 The highest average N-NO2- concentration was found in treatment 40-20-20 with a value of 0.6±0.66 mg L-1 while treatment 10-5-5 and 20-10-

10 were 0.27±0.33 and 0.52±0.61 mg L-1 A suitable range of nitrite for marine shrimp culture is below 1.0 mg L-1 (Boyd, 1998) Thus, NO2- concentrations in the present experiment did not cause any shrimp mortality TAN and N-NO2- concentrations among treatments are shown in Table 12

Table 12: TAN, Nitrite, and Phosphate for white shrimp reared in low salinity water in Experiment 2

Values represent the Mean ± Standard deviation (Min-Max) during experiment

In this experiment, NaH2PO4 was added to four treatments at levels 0, 5, 10, and 20

mg L-1 P-PO43- Therefore, average fluctuation of phosphate reading was between 0.11 and 14.86 mg L-1 The highest average P-PO43- concentration was found in treatment 40-20-20 with a value of 8.83±3.56 mg L-1 while treatment 10-5-5 and 20-10-10 were 4.33±0.69 and 6.42±1.27 mg L-1 P-PO43- concentrations in the present experiment did not cause any shrimp mortality P-PO43- concentrations among treatments are shown in

Table 12

4.2.1.3 Calcium (Ca 2+ ), Magnesium (Mg 2+ ), Potassium (K + ), and Mg:Ca ratios

Table 13: Potassium, magnesium, and calcium for white shrimp reared in low salinity water in Experiment 2

96.4±19.15 (71.9-115)

33.48±2.0 (30.7-36)

10-5-5

38.7±1.11 (37.3-40.2)

107.4±12.05 (95.3-121.8)

34.38±2.9 (30.7-38.2)

20-10-10

38.6±1.48 (37.4-41.0)

125.1±18.52 (105.8-150.6)

33.2±3.0 (29.2-36.4)

40-20-20

58.54±1.12 (57.0-60.0)

177.6±6.33 (171.5-188)

33.64±3.12 (30.3-38)

Values represent the mean ± standard deviation (min-max) during experiment

Calcium (Ca2+) concentrations were ranged from 29.2 to 36 mg L-1 during the experiment period Magnesium (Mg2+) was added to provide concentrations of 0, 10,

20, and 40 mg L-1 per tank to three replicate tanks per treatment, respectively Consequently, the average concentrations of magnesium were 96.4±19.15,

magnesium concentrations lead to increase hardness concentrations in the water The maximum hardness was recorded in treatment 40-20-20 (565 mgCaCO3 L-1)while the lowest one was in control group (256.5 mgCaCO3 L-1) (Table 14)

In this experiment, water was supplemented with 0, 5, 10, 20 mg L-1 potassium (K+) from potassium chloride (KCl) Potassium concentrations were recorded 33.16±0.94, 38.7±1.11, 38.6±1.48, and 58.54±1.12, respectively (Table 13)

Table 14: Hardness and Mg:Ca ratios for white shrimp reared in low salinity water in Experiment 2

Values represent the mean ± standard deviation (min-max) during experiment

In this study, magnesium was supplemented in the water Thus, ratios of Mg:Ca in water in treatments were higher that of control group In fact, the ratios of Mg:Ca in control, 10-5-5, 20-10-10, and 40-20-20 treatments were 2.86±0.42, 3.12±0.12, 3.75±0.25, and 5.30±0.31, respectively

4.2.2 Growth performance of white shrimp L vannamei

4.2.2.1 Molting intervals of white shrimp L vannamei

In the first molting, the molting interval was longest in 20-10-10 treatment (12.1±1.1 days), and shortest in 40-20-20 treatment (10.4±0.4 days) (Fig.5) In the second molting, average molting intervals were 10.8±0.5, 11±1.0, 10.4±0.4, and 10±0.3 days, respectively In general, the various Mg-K-P supplemented levels were not effect on molting interval of white shrimp No significant differences in molting interval among treatments were found (p>0.05)

One of study of Corteel et al (2012) reported that total molting intervals of white

shrimp L vannamei with weight of 2 g and 15 g were 5 days and 11 days, respectively

In addition, juveniles weighing 1-5 g, molting was every 4-6 days However, in

Corteel et al (2012) study, shrimp were reared in salinity of 35‰ In the present study, shrimp was reared in low salinity (2‰), thus molting intervals was longer compared to with shrimp cultured in 35‰

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Fig.5: Molting intervals of white shrimp (L.vannamei)

4.2.2.2 Weight gain of white shrimp after molting

After first molting, WG of shrimp in the 20-10-10 treatment showed significantly higher than those of other treatments (mean of 0.59±0.03 g) (p<0.05) (Fig.6) However, after second molting, WG in the 20-10-10 treatment was lowest compared to other treatments (p< 0.05) Weight gain of shrimp increased in control and 10-5-5 treatments with the mean of 0.58±0.05 and 0.6±0.1 g, respectively

a ab

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Fig.7: Mean weight of shrimp after each molting

4.2.2.3 Length gain of white shrimp after molting

After first molting, length gain of shrimp reared in the 20-10-10 treatment was 0.45±0.05 cm, significantly higher than that of other treatments (Fig.7) which ranged from 0.17±0.03 cm to 0.23±0.04 cm After second molting, length gain of shrimp in the 20-10-10 treatment was highest compared to other treatments (p< 0.05) but not significantly different with that of 40-20-20 treatment

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Fig.9: Mean length of shrimp after each molting

4.2.2.4 Growth performance of white shrimp at the end of experiment

mg L-1 could effectively improve growth performance of white shrimp

In general, WG and DWG of white shrimp (4.47±0.02 g) were higher than shrimp (PL15) in Experiment 1 while SGR was lower in all treatments In addition, Perez-Velazquez el al (2007) reported shrimp reared at 2‰ had significantly greater final weight (3.87 g) and weight gain (3.5 g) than animals kept at 35‰ (3.40, 3.04 g) or 50‰ (2.84, 2.47 g)