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 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
i

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

DANG MINH QUAN




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./m
2
. 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./m
2
(94.00%) was higher than at 600 ind./m
2
(89.44%),

900 ind./m
2
(82.30%) and 1200 ind./m
2
(80.89%), there was significant difference
(P<0.05). Rearing at 300 ind./m
2
,

snails reached highest body weight and shell height
(0.33 g and 11.54 mm) compared to 600 ind./m
2
(0,29 g and 10.29mm), 900 ind./m
2
(0.23 g and 9.84 mm) and 1200 ind./m
2
(0.21 g and 9.70 mm). At the stocking density
of 600 ind/m
2
,
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./m
2
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./m
2

. 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/m
2
) compare to
other treatments, but no significant difference (p>0.05). Results indicated that Pila
polita were reared at 600 ind./m
2
and fed with RB fulfilled the requirements on
interested aspects such as growth performance, survival, productivity and economic
effectiveness.

iii


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

iv

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

v


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 (SGR
weight
, %/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

vi

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 NO
2
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 NO
2
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


vii

LIST OF ABBREVIATIONS


DLG
Daily length gain
DWG
Daily weight gain
SGR
length
Specific growth rate in shell length

SGR
growth
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

1

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.

2

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.

3

CHAPTER II
LITERATURE REVIEW

2.1 Classification
Phylum: Mollusca
Class: Gastropoda
Order: Architaenioglossa
Family: Ampullariidae
Genus: Pila
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

4

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 (
SHELL

Surface
smooth to rough (growth
lines)
Shape
egg-shaped, ovoid to globose
Direction
right (dextral)
Whorls

round
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.

5

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

6

the high nutritional value of this alternative food source is high, this behaviour fits
well in their survival strategy (
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/m
2
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. ()
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/m
2
. Stocking density varied from 100-150 snails/m
2

and at 80 - 120 snails/m
2
if bigger snail size was selected. Snails were fed daily with
rice bran, cassava, sweet potatoes, vegetables, meat, or trash fish. Foods were fed

7

once a day with daily amount was 10% body weight of cultured snails
()
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/m
2
and 150 snails/m
2
.
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/m
2
showed higher growth, survival rate and economic efficiency
than stocking of 150 snails/m
2
.
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

8

period.
Le Van Binh et al. (2013) investigated the effects of different foods on the growth
and survival rate of Pila polita. The results showed that after 35 days of nursing
period, the survival rate of snails in rice bran diet (94.4%) was no significant
difference (P>0.05) from cassava powder (93.3%) or industrial pellet (93.7%).

Highest increased biomass was obtained when feeding snails with pellet (2,027%) and
significant difference compared to rice bran (727%) and cassava powder (992%).
Feeding with pellet, snail also reached highest body weight and shell height (0.71g
and 14.79mm) compared to feeding with rice bran (0.26g and 10.55mm) or cassava
powder (0.36g and 11.65mm). Snails were fed with pellet also presented highest feed
efficiency (723%) and that was significant difference compared to feeding with rice
bran (473%) or cassava powder (529%).
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

9

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.




10

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, NH
3
/NH
4
+
, NO

2
-
,
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.

11


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).

12


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

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



13

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:



14

Experiment 1: Effects of different stocking densities 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) was released at different the stocking densities as
follow:
+ Treatment 1: 300 snails/m
2
+ Treatment 2: 600 snails/m
2

+ Treatment 3: 900 snails/m
2

+ Treatment 4: 1200 snails/m
2


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.



15

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
pH
Once a week
Test kit
Alkalinity
Once a week
Test kit
NH
4
/NH
3

Once a week
Test kit

NO
2

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) = (W
final
– W
initial
)/t
Specific growth rate (SGR) in shell length
SGR
W
(%/day) = (ln (W
final
) – ln (W
initial
)) ×100/t
Daily length gain (DLG)
DLG (mm/day) = (F
inial

– L
initial
)/t
Specific growth rate (SGR) in body length
SGR
L
(%/day)= (ln(L
final
) – ln(L
initial
))×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 (%) = (N
t
/ N
o
) × 100
N

t
: Number of alive snails
N
o
: Initial number of released snails

16

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/(W
f
- W
i
)
m: amount of feed used
W
i
: snail weight at the beginning of experiment
W
f
: snail weight at the end of experiment
Productivity
P (g/m
2
) = W

final
× SR
Where:
W
final
: body weight of snails at the end of experiment
SR: survival rate