effects of different stocking densities and diets on the growth and survival rate of black apple snail (pila polita)

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES EFFECTS OF DIFFERENT STOCKING DENSITIES AND DIETS ON THE GROWTH AND SURVIVAL RATE OF BLACK APPLE SNAIL Pila polita By DANG MINH

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

EFFECTS OF DIFFERENT STOCKING DENSITIES AND DIETS ON THE GROWTH AND SURVIVAL RATE

OF BLACK APPLE SNAIL (Pila polita)

By

DANG MINH QUAN

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture Science

Can Tho, December 2013

CAN THO UNIVERSITY COLLEGE OF AQUACULTURE AND FISHERIES

EFFECTS OF DIFFERENT STOCKING DENSITIES AND DIETS ON THE GROWTH AND SURVIVAL RATE

OF

BLACK APPLE SNAIL (Pila polita)

By

DANG MINH QUAN

A thesis submitted in partial fulfillment of the requirements for

the degree of Bachelor of Aquaculture Science

Supervisor Assoc.Prof.Dr NGO THI THU THAO

Can Tho, December 2013

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

I would like to thank Assoc Prof Dr Ngo Thi Thu Thao and Mr Le Van Binh who have instructed me enthusiastically to finish this graduating thesis

For other valuable help and guide, thanks are extended to all my friends, Mr

Le Van Binh, Mr Nguyen Chi, Ms Nguyen Kim Cuong, Ms Tran Thi Be Gam, Ms Nguyen Khanh Linh and friends in AAP course 35

I wish to express my sincere gratitude to my advisor, Dr Duong Thuy Yen for her 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

ABSTRACT

This study consists of two experiments on Black apple snail Pila polita The

first experiment was aimed to evaluate the effects of different densities on the growth and survival rate There were 3 replicates in each treatment and densities as follow:

300, 600, 900, 1200 ind./m2 Snails with initial weight (0.03g) and shell height (4.5 mm) were reared in the composite tanks and fed with industrial pellet After 42 days, the survival rate at 300 ind./m2 (94.00%) was higher than at 600 ind./m2 (89.44%),

900 ind./m2 (82.30%) and 1200 ind./m2 (80.89%), there was significant difference (P<0.05) Rearing at 300 ind./m2,snails reached highest body weight and shell height (0.33 g and 11.54 mm) compared to 600 ind./m2 (0,29 g and 10.29mm), 900 ind./m2 (0.23 g and 9.84 mm) and 1200 ind./m2 (0.21 g and 9.70 mm) At the stocking density

of 600 ind/m2, snails also presented highest feed efficiency (644.4 %) and that was significant difference (p<0.05) compared to other treatments In results, snails were reared at density of 600 ind./m2 had a good growth performance, survival, productivity and economic effectiveness

The second experiment was conducted to evaluate the effect of different diets

on the growth and survival rate of snail Pila polita Snails were reared at density of

600 ind./m2 A triplicate experiment with 4 different treatments as follow: Rice bran

incubated with Bacillus subtilus (RB), RB + B subtilus supplemented directly into cultured tank, Industrial pellet powder incubated with Bacillus subtilus (IPB) and IPB + B subtilus supplemented directly into cultured tank After 42 days, survival rate of

treatment 2 (88.00±5.17%) were higher than treatment 1 (81.89±11.03%), treatment 3 (75.00±11.355) and treatment 4 (79.44±7.65%) but no significant difference (p>0.05) Treatment 1 reached highest body weight and shell height (0.20g and 9.51mm) compared to treatment 2 (0.19g and 9.14mm), treatment 3 (0.18g and 9.05mm) and treatment 4 (0.18g and 9.06mm), however there were significant differences (p<0.05) Treatment 2 had the lowest FCR value (0.36) but no significant difference (p<0.05) compare to other treatments Feeding with treatment 1 had the highest value in biomass growth rate (106.74%), FE (414.22%) and yield (82.14g/m2) compare to

other treatments, but no significant difference (p>0.05) Results indicated that Pila

polita were reared at 600 ind./m2 and fed with RB fulfilled the requirements on interested aspects such as growth performance, survival, productivity and economic effectiveness

TABLE OF CONTENTS

Acknowledgements i

ABSTRACT ii

LIST OF TABLES v

LIST OF ABBREVIATIONS vii

CHAPTER 1 1

INTRODUCTION 1

1.1 Introduction 1

1.2 Objectives of the study 2

1.3 Contents of research 2

CHAPTER II 3

LITERATURE REVIEW 3

2.2 Biological characteristics 4

2.2.1 External morphology 4

2.2.2 Habitat and distribution 4

2.2.3 Food and nutrition 5

2.2.4 Reproduction 6

2.3 Seed production and grow out 6

2.3.1 Seed production 6

2.3.2 Grow out 6

2.4 International and domestic research activities on Pila polita 7

2.4.1 International research activities 7

2.4.2 Domestic research activities 7

2.5 Probiotics in aquaculture 8

CHAPTER III 10

MATERIALS AND METHODS 10

3.1 Time and location 10

3.2 Materials 10

3.2.1 Equipment 10

3.2.2 Feeds 10

3.2.3 Water resource 11

3.2.4 Incubated feed making proceduce 11

3.3 Methods 13

3.3.1 Experimental design 13

3.3.2 Sampling and data collection 15

3.3.3 Determine the total density of bacteria, Vibrio and Bacillus sp in the tank 17 3.4 Statistical analysis 17

CHAPTER IV 18

RESULTS AND DISCUSSIONS 18

4.1 Effects of different stocking densities on the growth and survival of Pila polita 18

4.1.1 Water quality parameters 18

4.1.2 Growth of Black apple snail 21

4.1.3 Growth rates of snails 23

4.1.4 Biomass growth rate and feed efficiency of snail 24

4.2 Effects of different diets on the growth and survival rate of Pila polita 25

4.2.1 Water quality parameters 25

4.2.2 Total bacteria counts and density of Bacillus in different treatments 29

4.2.3.1 Growth rate in shell height 30

4.2.3.2 Growth rate in body weight 32

CHAPTER 5 35

CONCLUSIONS AND RECOMMENDATIONS 35

5.1 Conclusions 35

Effects of stocking density on the growth and survival rate of Pila polita 35

Effects of different diets on the growth and survival rate of Pila polita 35

5.2 Recommendations 35

REFERENCES 36

APPENDIX 41

LIST OF TABLES

Table 2 1 Characteristics of the genus Pila 4

Table 3.1 Physical and chemical parameter collection………….……… 15

Table 4 1 Mean values of environmental parameters during culture period 21

Table 4 2 The specific growth rate in shell length during experiment (%/day) 23 Table 4.3 The specific growth rate in snail body weight (SGRweight, %/day) 24 Table 4 4 Survival rate, biomass growth rate, Feed conversion ratio (FCR) in

different stocking densities 25 Table 4 5 Mean values of environmental factors in the treatments 28

Table 4 6 The Specific Growth Rate (%/day) of snail body weight during rearing

period 33 Table 4 7 Survival rate, biomass growth rate, feed conversion ratio (FCR) in the same snail treatments 34

LIST OF FIGURES

Figure 2 1 Snail from side view and abdominal view 3

Figure 3 1 Biosubtyl DL and Biosubtyl-II……….11

Figure 3 2 Rice bran incubated with Bacillus subtilus (RB) 12

Figure 3 3 Industrial pellet powder incubated with Bacillus subtilus (IPB) 12

Figure 3 4 Experimental system 13

Figure 4 1 Variation of temperature during experimental period.……… 18

Figure 4 2 Variation of pH during experimental period 19

Figure 4 3 Variation of TAN during the culture period 20

Figure 4 4 Variation of NO2 during the culture period 20

Figure 4 5 Variation of Alkalinity during the culture period 21

Figure 4 6 Variation of shell length of snails during the culture period 22

Figure 4 7 Variation of snail body weight during the culture period 23

Figure 4 8 Variation of temperature during experimental period 26

Figure 4 9 Variation of pH during experimental period 27

Figure 4 10 Variation of TAN during experimental period 27

Figure 4 11 Variation of NO2 during experimental period 28

Figure 4 12 Variation of a total bacteria counts in different treatments during 7 days 29

Figure 4 13 Variation of Bacillus density in different treatments during 7 days 30

Figure 4 14 Variation of average shell height of snail during experimental period 31

Figure 4 15 Variation of specific growth rate in shell height during experimental period 31

Figure 4 16 Total weight of snail in the treatments during experimental period 32

Figure 4 17 Variation of survival rate of snail during experimental period 33

LIST OF ABBREVIATIONS

DLG Daily length gain

DWG Daily weight gain

SGRlength Specific growth rate in shell length

SGRgrowth Specific growth rate in body weight

IPP Industrial pellet powder

RB Rice bran incubated with Bacillus subtilus (RB)

IPB Industrial pellet powder incubated with Bacillus subtilus (IPB)

SL Shell length

W initial Weight at initial time of experiment

L initial Length at initial time of experiment

W final weight at the end of experiment

L initial length at the end of experiment

t Experiment duration (days)

SR Survival rate

Nt Number of alive snails

No Initial number of released snails

FE Feed efficiency

FCR Feed conversion ratio

CHAPTER 1 INTRODUCTION 1.1 Introduction

In recent years, with the development trend of the world economy, the domestic aquaculture industry has been developing rapidly in both quantity and quality, as well

as expanding the model culture Beside providing food, aquaculture products are also exported with high economic value, so aquaculture have become a key sector of the economy of Vietnam Vietnam has many advantages and suitable conditions for aquaculture development With a coastline of 3620 km, 112 of the river system of rivers, canals, lagoons and dense, large water surface field is a favorable condition to develop aquaculture To promote the advantages which require investment in research planning and supply of seed for the needs of the farmers

In freshwater aquaculture recent years has become a professional manufacturer to bring high economic efficiency Freshwater aquaculture training products help improve people's lives and contribute to poverty reduction In addition to the traditional freshwater species such as common carp, silver carp, catfish, a species as

black apple snails (Pila polita) is relatively new, but very promising because it is

relatively high economic value The dish is made from Black apple snails with delicious quality, nutritious (containing 11.9% proteins; 0.7% lipid; vitamins B1, B2, PP; the mineral Ca, P; provide energy of 86 calo/100g meat), amino acids and in

particular contain unsaturated healthy fats (Do Huy Bich et al, 2003)

Due to the current supply of Black apple snails mostly from by wild-caught therefore it may be not stable, also it will increase the fishing pressure lead to a decline of biodiversity in fresh waters Currently, a few studies conducted in breeding Black apple snails with seed collecting from the wild The results are quite satisfactory, and could bring high economic efficiency for the farmers On the other hand, this study also contribute a solution to reduce the fishing pressure and conserve natural resources of local species Based on research and practical demands, this study

on “Effects of different stocking densities and diets on the growth, and survival rate of

black apple snail Pila polita” was be carried out

1.2 Objectives of the study

To find out the appropriate foods and stocking density for rearing Black apple

snails These results could contribute to improve culture technique of Pila polita

1.3 Contents of research

Experiment 1: evaluate the effect of different stocking densities on the growth and survival rate of Black apple snails

Experiment 2: evaluate the effect of different diets on the growth and survival rate

of Black apple snails

CHAPTER II LITERATURE REVIEW

Specific name: polita

Scientific name: Pila polita

English Name: Black Apple Snail

Vietnamese name: Ốc bươu đồng, Ốc nhồi

In the world, so far 23 species of Pila.sp has classified, including: P africana,

P Africana martens, P ampulacea, P ampullacea, P angelica, P cecillei, P

congoensis, P conica, P globosa, P gracilis, P leopoldvillensis, P letourmenxi, P luzonica, P occidentalis, P ovata, P pesmei, P pesmi, P polita, P saxea, P scutata,

P speciosa, P virens, P wernei

Figure 2 1 Snail from side view and abdominal view

2.2 Biological characteristics

2.2.1 External morphology

Large snails, glossy shell surface, blue yellow or brown colour outside, the inside have light violet colour The number of whorls is 5.5 - 6, slightly bulging and shallow twisted grooves Long narrow operculum with the width is at half height, sharp top shell The last spire is large, up to 5/6 of shell height while the spirals above are small

Table 2 1 Characteristics of the genus Pila (http://www.applesnail.net/content/pila.htm)

SHELL

Surface smooth to rough (growth

lines) Shape egg-shaped, ovoid to globose Direction right (dextral)

Shell-opening (aperture) oval to egg-shaped Umbilicus wide, narrow to closed

OPERCULUM

Colour yellow, dark brown to almost

black, with or without spiral bands

Structure corneous outside, calcified

inside BODY

Head (cephalic) tentacles Long Labial tentacles Long Breathing siphon Medium EGGS

Colour grey-yellow to grey, with

dark spots Position above the waterline

2.2.2 Habitat and distribution

Dillon (2000) reported that Pila polita are present in fresh waters in Indochina,

Indonesia, China, Thailand and Vietnam They live in ponds, fields, plains and midlands

When moving, snails open operculum, spread the abdominal muscles as flexible blade on the bottom or on the wall, and secretes a mucous layer to reduce friction While moving the head protruding, mouth lobe is in the middle and two siphon trunks

The feet withdraw into the shell when operculum closes and stretching when moving,

at this time operculum fold on the back side Snails usually float to the surface to breathe, when there is noise, snails immediately withdraw into the shell and drive to the bottom In hot or cold weather, they float to the surface Snails have both gills and lungs so they can live in water and on land

Being as fresh water snail, but some Ampullariids species may be able to tolerate

at low levels of salinity (Prashad, 1925; Hunt, 1961; Fujio et al., 1991; Santos et al.,

1987), however they generally do not live in brackish waters Most species are amphibious, able to spend significant lengths of time out of water for breathing air

Many species, especially Pila, Pomacea, Marisa and Lanistes, inhabit slow-moving

or stagnant waters in lowland swamps, marshes, ditches, lakes, and rivers (Pain, 1950, 1960; Andrews, 1965; Robins, 1971; Louda and McKaye, 1982; Keawjam, 1986)

2.2.3 Food and nutrition

Black apple snail are not selective feeding and eat almost everything available in their environment In general, they prefer soft and digestible vegetation Tougher plants and algae are consumed as long as they are able to grasp pieces of with their radula (rasp tongue) When there is not enough food available in the water, Black apple snails can profit from their amphibian life style to leave the water in search for food

An interesting strategy used by Black apple snails to attain food is exhibited when the food is floating on the surface (surface film feeding) In that case the snail crawls

to the surface and forms a foot-funnel in to trap particles from the surface To attract more floating food, the snail makes the same movement with its foot as it does for walking with the front part of the foot The middle part and the tail of the foot, the snail remain attached to the side or an object near the surface Once the funnel is filled, the snail brings its head in the funnel and eats the collected material Behaviour is known as ciliary feeding

Black apple snails are opportinustic and even consume all kinds of dead animals like dead fish, frogs, crustaceans and insects and eggs (fish, frogs, snails etc.) Since

the high nutritional value of this alternative food source is high, this behaviour fits well in their survival strategy (http://www.applesnail.net/content/ecology.php)

2.2.4 Reproduction

Ampullariids are dioecious, internally fertilizing and oviparous (not reciprocally –

fertilizing hermaphrodites as stated by Chang, 1985) Species of Pila have been

reported to change sex (Keawjam, 1987; Keawjam and Upatham, 1990) The sex change is from male to female (protandry) and takes place during aestivation The

larger size of females in Pila has therefore been attributed to continuing growth

following this change

2.3 Seed production and grow out

2.3.1 Seed production

Ponds with soft muddy bottoms, organic humus, average water levels around 0,5m and slightly flow are suitable for seed production Ponds were fertilized with chicken manure, dung - cow mixed with chopped straw (1/3) Fertilizing the snail pond was done before stocking at least 3 days Stocking density is 15-20 snails/m2 with male/female ratio is 1:1 Broodstock snails were released before breeding season and baby snails could be collected after 10-15 days Care should be taken to avoid broken snail shells (http://www.kinhtenongthon.com.vn)

2.3.2 Grow out

Snails are stocked at rice field, with 0.7-1m in the ditch and 0.2-0.3m in the platform Normally, snails were integrated culture in ditches, ponds, fields with fish Lotus were planted to cool down the temperature and to be shelters for snails Water would be better with slightly flow, must not contaminated with pesticides or rich in organic matter Ponds should be fertilized with chopped straw and manure before stocking 10 days at a rate of 2 kg/m2 Stocking density varied from 100-150 snails/m2and at 80 - 120 snails/m2 if bigger snail size was selected Snails were fed daily with rice bran, cassava, sweet potatoes, vegetables, meat, or trash fish Foods were fed

once a day with daily amount was 10% body weight of cultured snails (http://www.kinhtenongthon.com.vn)

2.4 International and domestic research activities on Pila polita

2.4.1 International research activities

Dillon (2000) studied the distribution of snails Pila polita, the authors identified

Pila polita commonly distributed in Indonesia, China, Thailand and Vietnam The

author also reported that Pila polita lived in the farm ponds and midland plains

Keawjam (1986) and Thaewnon-ngiw et al (2003) studied on medical effects of

Pila polita The authors mentioned that the snail is one of eight species of freshwater

snails which have an important role in medicine It is used to treat skin diseases for local community in southern Thailand

2.4.2 Domestic research activities

Nguyen Thi Dat (2010) studied on the effects of different densities and feeds on

the growth and survival rate of the snail Pila polita in grow-out period Snails were

fed with cassava leaves, homemade feed (40% rice bran, 20% corn, 10% lighter fish meal and 30% soybean meal) Stocking density was 100 snails/m2 and 150 snails/m2 Results showed that feeds and densities significantly affected the growth and survival rates of the snails Vegetables combined with homemade diets resulted in higher growth performance, survival rate and economic efficiency Moreover, stocking with density of 100 snails/m2 showed higher growth, survival rate and economic efficiency than stocking of 150 snails/m2

Nguyen Thi Binh (2011) studied on reproductive and biology characteristics of the

snail Pila polita The results showed that Pila polita was dioecious species and the

ratio of male and female was 1:1.67 Snails were often pairing and mating several times before spawning and female snails laid eggs at night time The main spawning season was from April to June with the mature proportion from 62.2 to 93.3% The authors also found that stocking snails in the muddy bottom would be better than others and the survival rate varied from 88.15 to 90.93% after 28 days of cultured

2.5 Probiotics in aquaculture

A widely accepted definition is taken from Fuller (Fuller R, 1987), who considered that a probiotic is a cultured product or live microbial feed supplement, which beneficially affects the host by improving its intestinal (microbial) balance The important components of this definition reflect the need for a living microorganism and application to the host as a feed supplement (Sahu, 2008) Moriarty (1998) and

Rengpipat et al (1998) indicated that probiotics may prevent the luminous bacteria -

Vibrio species effectively Intervention mechanism may be a combination of

competition between bacteria and different antibiotic compounds by Bacillus spp

created According to some recent researches in aquaculture, the mechanism of probiotics can be divided into following aspects: (1) production of inhibitory compounds, (2) competition for nutrients maintenance, energy, shelter for harmful bacteria, (3) enhance the immune response, (4) improve water quality (Pham Thi Tuyet Ngan, 2010)

Several studies have conducted to investigate the effects of probiotics on mollusc Ngo Thi Thu Thao & Pham Thi Tuyet Ngan (2011) studied the effect of probiotics on

larvae of Balylonia areolata Results showed that treatment with probiotics

supplement, the survival and growth rate was higher than a control treatment Ngo Thi

Thu Thao et al (2012) studied on the effect of different applied methods of the probiotics in juvenile of clam Meretrix lyrata The results showed that

supplementation of probiotics into algae biomass and added directly into the environment led to faster growth in weight and length compared to non-probiotics treatment (p<0.05) Results from these studies showed that the probiotics application has been effective in the nursery as well as in aquaculture In recent years, the Ministry of Fisheries of Vietnam has allowed the application of microbial products, farmers have become familiar with the biological products and get good results However, it should be a comprehensive assessment of economic efficiency and probiotic using methods

CHAPTER III MATERIALS AND METHODS

3.1 Time and location

Experiments were be carried out from May to December, 2013 at Department

of Coastal Aquaculture, College of Aquaculture and Fisheries, Can Tho University

3.2 Materials

3.2.1 Equipment

Tanks: 24 square tanks (60cm × 60cm);

Electronic balance (0.01g readability), vernier caliper, thermometer, aeration;

pH, NH3/NH4

+

, NO2

, and alkalinity test kits; subtracts (water lettuce, Pistia

-stratiotes)

3.2.2 Feeds

Pellet feed (18% protein), fine rice bran

3.2.3 Probiotics

Biosubtyl DL: contain Bacillus subtilus and Lactobacillus acidophilus

Biosubtyl-II: contain Bacillus subtilus

Figure 3 1 Biosubtyl DL and Biosubtyl-II

3.2.4 Water resource

Fresh water is taken from fish ponds at College of Aquaculture and Fisheries, Can Tho University Water is pumped to the settling tanks for 2-3 days and then filtered through a plankton net (50 µm mesh size) into the incubation tank

3.2.5 Incubated feed making proceduce

Firstly, well mix 1kg fine bran (pellet feed) and 5 Biosubtyl-II packages together, then put 500gr molasses and little amount of water to dissolve the sugar and create adhesion for the mixture After the mixture is mixed well, exposing the mixture

at room temperature until completely dry Finally, crushed and filtered through a mesh to a fine powder (Figure 3.2, 3.3)

Figure 3 2 Rice bran incubated with Bacillus subtilus (RB)

Figure 3 3: Industrial pellet powder incubated with Bacillus subtilus (IPB)

3.3 Methods

3.3.1 Experimental design

This study consisted of 2 experiments, Experiment 1: Selecting the best

stocking density for nursing juvenile snails and Experiment 2: Selecting the best diet

for nursing juvenile snails The best stocking density from Experiment 1 would applied for second experiment

The total of 12 composite tanks (60×60 cm in dimension and 200 liters in capacity) was used for nursing snails Tanks was cleaned carefully before use (Fig 3.4)

Figure 3 4 Experimental system Water volume in tank maintained at about 30 liters (12-15 cm water depth) Two rectangular feeding trays (20 × 15 cm) were laid at the bottom of each tank All

of experiments were set up in-door with aeration continuously Specific methods and management for each experiment were described as follow:

Experiment 1: Effects of different stocking densities on the growth and survival of

Snails were fed 2 times per day (7:30 and 17:00) with the quantity of 3-5% of snail biomass in each tank Water was renewed weekly with the ratio of 50% total volume in each rearing tank and added probiotics (Biosubtilus DL) twice a week Aeration was supplied continuously, but no substrate was laid in the rearing tank This experiment was run for 42 days

Experiment 2: Effects of different diets on the growth and survival of Pila polita

This experiment includes 4 treatments and 3 replicates were run for each Snails (Shell Length:~5.0-9.0 mm) were released at the best stocking density from Experiment 1 with different diets as follow:

+ Treatment 1: Rice bran incubated with Bacillus subtilus (RB)

+ Treatment 2: RB + B subtilus supplemented directly into cultured tank + Treatment 3: Industrial pellet powder incubated with Bacillus subtilus (IPB) + Treatment 4: IPB + B subtilus supplemented directly into cultured tank Substrates (water lettuce, Pistia stratiotes) and probiotics (Biosubtyl-II) were

used and replaced weekly when renewing water Snails were fed 2 times per day with 3-5% of biomass Aeration was supplied continuously, water exchange was done as Experiment 1 This experiment run for 42 days

3.3.2 Sampling and data collection

The environmental parameters and analytical methods are shown in Table 3.1 Table 3 1 Frequency and methods to observe the environmental parameters Water parameters Sampling frequency Equipment Temperature 2 times per day (7 am-2 pm) Thermometer

Alkalinity Once a week Test kit

At the beginning and 7 day intervals during the experiment, numbers of snails

in each tank was be counted for checking the survival rate

By weekly, shell height and body weight of 50 snails/tank was be measured and weighed to determine the growth rate Total weight of alive snails in each tank will be also weighed to obtain the biomass data

Feed used and consumed was be collected daily to calculate FCR (feed conversion rate) Following formulas was be applied to calculate the results:

Daily weight gain (DWG)

DWG (g/day) = (Wfinal – Winitial)/t Specific growth rate (SGR) in shell length

SGRW (%/day) = (ln (Wfinal) – ln (Winitial)) ×100/t Daily length gain (DLG)

DLG (mm/day) = (Finial – Linitial)/t Specific growth rate (SGR) in body length

SGRL (%/day)= (ln(Lfinal) – ln(Linitial))×100/t Where:

W initial, L initial: weight and length at initial time of experiment

W final, L initial: weight and length at the end of experiment t: Experiment duration (days)

Survival rate (SR)

SR (%) = (Nt / No) × 100

Nt: Number of alive snails

N : Initial number of released snails

Biomass

Biomass (g/tank) = Body weight (g/snail) × Number of snails in tank Biomass increase rate (%) = (Biomass increase/Biomass initial) × 100 Feed conversion rate (FCR)

FCR = m/(Wf - Wi)

m: amount of feed used

Wi: snail weight at the beginning of experiment

Wf: snail weight at the end of experiment Productivity

P (g/m2) = Wfinal × SR Where:

Wfinal: body weight of snails at the end of experiment SR: survival rate

3.3.3 Determine the total density of bacteria and Bacillus sp in the tank

Probiotics based on Bacillus subtilus (107- 108 CFU/g) was added weekly to the nursing tank with the quantity of 100 mg/L to the cultured tank or 100 mg/g to the food After adding probiotics, water samples were collected on day 1, 3, 5, 7, and sampling was done continuously in 2 weeks Water samples were collected at the bottom of the tank then transfer into Falcon tubes (around 50 ml for each tank) and stored in cool condition if needed Microbiological analysis were conducted at the same day This technique was applied only for the second experiment

Method of determining the density of bacteria

1 Prepare serial dilutions of the broth culture as shown in the figure from a previous lab exercise (Isolation of Pure Cultures) Be sure to mix the nutrient broth tubes before each serial transfer Transfer 0.1 ml of the final three dilutions (10-5, 10-6,

10-7) to duplicate nutrient agar plates, and label the plates

2 Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrient agar plates until the medium no longer appears moist Return the spreader to the alcohol

3 Repeat the flaming and spreading for each of the remaining five plates

4 Invert the six plates and incubate at room temperature until the next lab period (or ~ 48 hours, whichever is the shortest) Only plates with 30 to 300 colonies are statistically valid

3.4 Statistical analysis

Data were analyzed for mean value, standard deviation by using Excel software, and Duncan test (One way ANOVA analysis, SPSS 16.0) to compare the significant different among treatments at p<0.05

CHAPTER IV RESULTS AND DISCUSSIONS

4.1 Effects of different stocking densities on the growth and survival of Pila polita

4.1.1 Water quality parameters

Mean values of environmental parameters were illustrated in Table 4.1 The average temperature ranged from 27.54oC to 29.76oC during the experience

Figure 4 1 Variation of temperature during experimental period

pH levels in the experiment were in the range of 8.04 – 8.22 The highest pH presented in treatment 2 (8.22±0.04) which no significant difference to treatment 3 (8.15±0.06) Besides, between treatment 3 and treatment 4 were neither significant difference with 8.11±0.02 and 8.04±0.04, respectively