26

Materials and Methods

Experiments for the present study were carried in commercial brackish

water shrimp ponds located near Gangapatnam, Nellore district, Andhra

Pradesh, India. An estuarine canal is passing near the ponds with continuous

supply of water throughout the year. All the ponds average size goes 0.5 hac

and rectangular in shape with clay loamy soil suitable for semi intensive type

of culture. Infrastructure facilities such as electricity, road, Water supply and

skilled labor were available for the ponds. Pump house with three 3H.P motors

and Generator (15KW) were also arranged in the farm area. Ponds were

designed and constructed with strong dykes having 1.5m avg. height to retain

min 1.0m avg. Water inlet system designed with PVC pipelines. Filter bags and

mesh were tied to the inlets for preventing the entry of pest and predators. 1.0m

width of sluice supported with wooden plank shutters were also constructed to

all the ponds for daily water exchange and draining. Paddle Wheel aerators

were placed in each pond. One pond was used as reservoir/treatment pond.

Before stocking with post larvae all the experimental ponds were treated

with pre-stocking management. Initially pond bottom was ploughed and top

soil was removed upto a depth of 10 cm for eliminating any black soil

deposition. Later it was allowed for complete drying. After 1 week pH of the

soil was tested for liming. Quick lime (Cao) and Dolomite (Ca Mg (Co3)2) were

uniformly distributed at the pond bottom and dykes at the rate of 1.5ton/hac

and 1ton/hac respectively to get the soil pH around 7.5. Organic carbon and

available Phosphorus and Nitrogen in the soil were also tested for further

fertilization. 10 kg bleaching powder was applied for preventing the pest and

predators. Raw cow dung 200 kg, Urea (5 kg) and triple superphosphate (20

kg) were also applied in each pond when the level of water was around 80cm.

Then all the ponds were completely filled. Transparency was checked with

Secchidisc before stocking the post larvae. This was common to all the

experiments in the present study.

27

Hatchery produced seed (Cp aqua. Pvt., Ltd., Nellore) were used in the

present study. Post larvae with an average size of 0.67g body wt. were used for

stocking in the experimental ponds. All the post larvae were equal in size and

free from disease. Before packing the post larvae were subjected to stress test

by 50% reduction in salinity and 100ppm formalin treatment to ascertain the

seed quality. Seed were also confirmed negative for Taura syndrome and White

spot syndrome virus in two step PCR assay. Seed were brought in oxygenated

poly ethylene bags filled 1/3 with filtered sea water and stocked @500 no/lit.

Thermocole boxes were used to prevent stress during transportation. Stocking

was done at early hours to avoid temperature stress. All the post larvae were

acclimatized before stocking into the ponds. All the bags were kept in pond

water and slowly water was allowed to mix in the bags for a period of 30

minutes to adjust with the pond temperature After the completion of

acclimatization Post larvae were released. Aeration was given with paddle

wheel aerators throughout the culture in all the experimental ponds. In first and

second experiments 10% water exchange was done for every fifteen days.

Shrimp were fed with supplemented feed twice a day morning 7am and

evening 5pm for the first week and later four times a day at 6am, 10am, 3pm

and 7pm. Crumble type feed was used in the first month of culture upto a size

of 2g. avg. body weight. Pellet type feed was used for the remaining period of

culture. Feeding rate was managed at 15% of the shrimp body weight for the

first month. Later it was maintained at 8% in the second month, 4% in the third

month and fourth month. Check trays were used for observing the daily feed

consumption of shrimp. Eight check trays were used in each pond to observe

the feed wastage. Sampling was done at every fortnight by Seine net for first

fifteen days and through cast net for the remaining culture period. Total no of

individuals and the average body weights were recorded in each sampling. Five

hauls were made in each pond for studying the growth performance of

L.vannamei with each sample containing 50-70 animals.

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Plate-3.1

Fig.1: Nauplius of L.vannamei

Fig.2: Metanauplius of L.vannamei

29

Plate-3.2

Fig.1: Zoea-1 of L.vannamei

Fig.2: Zoea-2 of L.vannamei

30

Plate-3.3

Fig.1: Mysis of L.vannamei

Fig.2: Post larvae of L.vannamei

31

In the present study three experiments were carried for knowing the

survival and growth performance of shrimp L.vannamei with different stocking

densities, different feeds and probiotics. In the first experiment post larvae

stocked at a density of 20pcs/m² in pond (P1), 30pcs /m² in pond (P2), 40pcs

/m² in pond (P3), 50pcs /m² in pond (P4) and 60pcs /m² in pond (P5). In this

experiment only one type of feed was given to all the pods (Commercial feed).

Survival and growth performance of L.vannamei at different densities were

studied in this experiment.

In the second experiment post larvae were stocked at 45pcs/m² in all

the ponds. Three different feeds were used in this experiment. One is

Commercial feed (F1) and the other two were locally formulated feeds. Feed

(F2) is formulated by the available animal ingredients and Feed (F3) is

formulated by the plant ingredients. The animal ingredients and plant

ingredients were given (Table 3.1 and 3.2). The survival, growth performance

and biochemical analysis were studied in this experiment.

In the third experiment ponds were treated soil and water probiotics

Super Ps (5lit/0.5ha.) and Super biotic (5kg/0.5ha.) (Cp aqua. Pvt.Ltd,

Chennai). Water probiotic (Super biotic) was applied 5kg after 10hr

fermentation in 200liter water and broadcasted throughout the pond. Soil

probiotic super Ps was mixed with sand and broadcasted throughout the pond.

Feed probiotic (UB probizyme, Unique biotic Ltd. Hyderabad) was supplied

along with the formulated feeds (dose at 10g/kg). The application and dosage

of probotic in treatment ponds were followed according to the manufacturer’s

instructions. In this study shrimp survival, growth performance and

immunological studies were carried under the influence of probiotic

application.

Growth performance was studied by different growth parameters.

Average daily growth (ADG), Specific growth rate (SGR) and Feed conversion

ratio (FCR) was calculated and statistically analyzed (ANOVA). Final weight

32

gain%, Survival rate (SR) %, total feed consumption and total yield (Kg) were

estimated. The following growth parameters were used for studying the growth

performance of L.vannamei.

Growth parameters: Final weight (g) a. Average daily gain (g) = ------------------------------------ Duration of culture (days) Final Weight (g) – Initial Weight (g) b. Final Weight Gain % = --------------------------------------------- × 100 Initial Weight (g) Ln (Final weight (g) ) - ln (Initial weight (g) ) c. Specific growth rate = --------------------------------------------- × 100 Duration of culture (days) No of shrimp harvested d. Survival rate % = -------------------------------------------- × 100 No of shrimp stocked Total feed fed (dry wt.) (kg) e. Feed conversion ratio = ----------------------------------------------- Total yield (kg)

At the end of the second experiment shrimp whole body composition

were estimated for biochemical analysis of shrimp digestion. After harvesting

shrimp samples were collected from all the experimental treated and control

ponds. They were properly cleaned with distilled water and dried in hot air

oven at 60˚c for 48hr’s or until dry matter with a constant weight is reached.

The dried samples were taken and analyzed for Moisture, Crude protein, Crude

fat and Ash. Moisture was determined by Oven-drying at 105oC for 24h.

Crude protein was analyzed by the Kjeldahl method after acid digestion. Crude

lipid was analyzed by the other extraction method by Soxhlet system (AOAC,

1990).

In the third experiment Immunological studies were carried with

Haemolymph to know the effect of probiotic on shrimp health. Sampling was

done at the end of the experiment to compare between the control and probiotic

33

treated shrimp of three different fed ponds. Haemolymph was extracted from

the rosrtal sinus using specially designed sterile capillary tubes with diameter

of 0.5mm, pre-rinsed with anticoagulant. Anticoagulant was prepared from

0.01M Tris HCl, 0.25 M sucrose, 0.1 M trisodium citrate, Double distilled

water. They were autoclaved and adjusted to pH 7.6 (Song and Hsieh, 1994)

and transferred to a sterile micro centrifuge tube with cooled anticoagulant.

Total haemocyte count (THC) was made using a Neubauer improved

haemocytometer and expressed as THC/ ml haemolymph. Phenoloxidase (PO)

activity was measured spectrophotometrically by using L-3, 4-

dihydroxyphenylalanine (L-DOPA) as the substrate (Soderhall, 1981). The

dopachrome formed was measured at 495 nm and phenoloxidase activity was

then expressed as the increase in absorbance per min per 100µl haemolymph.

Respiratory burst activity of hemocytes was measured spectrophotometrically

(Song and Hsieh, 1994) using nitroblue tetrazolium (NBT) as the substrate.

Total protein in the Hemolymph was estimated as per Lowery et al. (1951)

using bovin serum albumin as standard after precipitating the hemolymph with

80% ethanol.

Hydrological studies:

Physico-chemical parameters were studied in all the ponds of the three experiments. They were measured by the standard methods and field instruments. Water temperature was measured in the pond itself by using a standard centigrade thermometer. Field test instruments were used to analyze water pH (Digital mini – pH meter, model 55) and dissolved oxygen (YSI-58). Transparency was measured in terms of light penetration method using secchidisc (Boyd, 1990). Salinity was measured with refractometer (Japan). Water samples were taken weekly for analyzing the other physico-chemical parameters in all the ponds using standard methods (APHA, 1989). Total ammonia (APHA, 1989), Nitrate – Nitrogen (Boyd, 1984), and total alkalinity & Hardness (APHA, 1989) were analyzed by the standard protocols.

Sample collection: Water samples were taken randomly from ponds weekly and

sampler from at least five spots in each experimental pond between 9.00am to

34

4.30pm at a depth of 30cm below the water surface. The samples were mixed

together in a plastic container and analyzed for water parameters.

Analytical methods used for calculation of other parameters.

Total ammonia concentration was measured by Hach comparison

apparatus following the method reported by APHA, (1989), and the Deionized

ammonia (NH3) was calculated from total ammonia according to Boyd (1990).

Nitrate-nitrogen was measured by phenoldisulphonic acid method according to

Boyd (1984). Colour readings were measured with the spectrophotometer

(Milton roy 21D model) at 410 nm wave length. Total alkalinity and total

hardness were measured by titration according to APHA (1985).

Experimental feeds:

Feed1 (F1): The commercial feed selected for the experiments in the present

study was supplied by the company Cp Aqua. Pvt., Ltd., Chennai. The feed

was used in crumble form for the first month and pellet form in the next three

months. Ingredients of the feed was Fish meal, Shrimp head meal, Squid meal,

Soya bean, Cod liver, broken rice, wheat flour, cholesterol, phospholipids,

vitamins and minerals.

Feed (F2): It was prepared locally from the available ingredients of animal

based protein i.e Squilla meal, Shrimp meal, Chicken liver, Snail foot, Earth

worm meal, Cholesterol, Vitamins and minerals.

Feed (F3): It was also prepared locally from the available ingredients of plant

based protein i.e Groumd nut cake, Heated soya bean, Corn gluten, Pea meal,

Rice bran, Wheat flour, Corn starch, Soy oil, Cholesterol, Vitamin and

minerals.

Equipments and technical procedure used in the feed preparation:

1. Weighing scale 2. Sieve 3. Grinde 4. Mixer 5. Steamer 6. Pelletiser 7. Oven

35

All the ingredients used in the feeds were properly dried and made into

homogenized mixture by grinding into a particulate size. All the powdered

ingredients were properly weighed and mixed according to the selected feed

formulae. Then the ingredients were steamed with water in a steamer for about

fifteen minutes for improving the digestibility of the feed and also to destroy

the microorganisms.Then the cooked material was cooled and mixed with

vitamin& minerals.Finally feed was extruded through a Pelletiser and sundried.

Table: 3.1 Animal based ingredients of feed2 (F2)

Table: 3.2 Plant based ingredients of Feed3 (F3)

Ingredient Percentage

Ground nut cake 35%

Heated Soya bean 22.70%

Corn gluten 15.20%

pea meal 8.50%

Rice bran 8.20%

Wheat flour 4.50%

Corn starch 0.9%

Vitamin & mineral mix 2.80%

Soy oil 1.30%

Cholesterol 0.90%

Ingredient Percentage

Squilla meal 28.50%

Shrimp meal 27.60%

Chicken Liver 21.50%

Snail foot 13.86%

Earth warm meal 11.86%

Vitamin & mineral mix 2.40%

Cholestrol 1.20%

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Table - 3.3: Proximate composition of experimental feeds

Nutrients Feed (F1)

(commercial)

Feed (F2)

(Animal based)

Feed (F3)

(plant based)

Crude protein 37.29% 36.39% 36.15%

Total lipid 5.23% 6.5% 7.2%

Fibre 4.57% 4.12% 4.42%

Moisture 12.25% 18.72% 19.20%

Ash 13.71% 15.95% 16.12%

Probiotics Soil probiotic:

Super Ps – For bottom soil quality management 10liltre of Supre Ps was mixed in 50kg dry sand and broadcasted throughout the pond during morning hours with an interval of 15 days.

Water probiotic:

Super biotic – For water quality management 1.5kg of super biotic mixed with 200litre water and fermented under aeration for 8hr’s. It was broadcasted throughout the pond during morning hours in an interval of 15 days.

(Soil and water probiotic were supplied by the Cp aquaculture Pvt. Ltd.)

Feed probiotic:

UB probizyme - It is an unique blended probiotic with enzymes, vitamins and mineral complex mixed and given along with the feed every day at 10g/kg feed.

37

Composition: 4.3×105million CFU/kg (Lactobacillus sporogenes, Lactobacillus acidophilus, Bacillus Subtillis, Bacillus Licheniformis, Saccharomyces cerevisiae )

(Feed probiotic was supplied by UNIQUE biotec Pvt. Ltd.)

Harvesting

Shrimp were harvested after completion of 120 days of culture. One day before harvesting all feed inputs was stopped. One third of water from each pond was drained through outlet just before catching. Two third of the quantity was collected through outflow and remaining was handpicked. Shrimp were immediately transferred to the cleaning tubs provided near the pond. They were rinsed and chilled before packing. Random samples were collected during the weighing process for determining the individual weight. Mean final weight, were calculated by using the Individual weights. After quantifying the biomass mean final yield, feed conversion ratio and survival were calculated. Specific growth rate, average daily gain and Final weight gain % were determined using the standard formula.

Statistical analysis

To know the significant difference between the results Student t-test

(Fisher’s) and ANOVA were applied. Student t-test was applied for comparing

single factor and ANOVA was used for comparing multiple factors. All the

Data were expressed in the mean ± standard error. The results of Average daily

gain (ADG), Specific growth rate (SGR) and feed conversion ratio (FCR) were

subjected to ANOVA analysis. Total haemocyte count (THC), Phenoloxidase

(PO) value and Nitroblue tetrazolium (NBT) were also analyzed using one way

analysis of variance (ANOVA) and Duncan’s multiple comparison of the

means using SPSS 10.0 for Windows. According to Steel and Torrie (1980) the

significant differences among treatments were performed using f- factor

analysis at a level of P ≤ 0.05 significance.

38

Survival and Growth performance of Pacific white Shrimp Litopenaeus

vannamei (Boone1931) Under Different Stocking Densities

Introduction

With increased population and diminished natural fishing resources

supply of aquatic products have limited for the human consumption around the

world. In many Asian countries culture of fish and shrimp got scope for aquatic

production. Due to various reasons aquaculture is growing faster and gaining

importance in our country. Feed is one of the major components of production

expenditure, representing up to 60% of variable cost (Hepher, 1988; Tacon et

al., 1998). The success of its culture is solely depends on survival rates and

average weight of shrimp that it attains at the time of harvest. In aquaculture

“stocking density” should denote the concentration of which organisms are

initially stocked into a system. However, it is generally used to refer to the

density of organisms at point of time. It is considered to be one of the important

factors that affect on organisms growth, feed utilization and grass production.

The proper utilization of the space for the maximum production through

intensive culture can improve the profitability of the shrimp intensification.

Shrimp intensification by increasing stocking density is also found suitable to

overcome the problems of land shortage. On the other hand several studies

have indicated that on inverse relationship between the stocking density and

growth of shrimp (Krishna, 2006; 2009).

Survival and growth of the organisms are known to be influenced by the

availability of right type of food in right concentrations. Proper management of

nursery and rearing ponds involves the providing of growing larvae, juveniles

and adults with right kind of natural and supplementary food for right time

(Krishna, 2006). In practice, the densities at which farmers keep their stock are

based on the experience and institution, with codes of practice and hand books

being used as guide. Information regarding effect of stocking density of the

shrimp performance during intensive culture is limited, inconsistent and some

time controversial.

39

In recent years aquaculture intensification has became a common practice throughout the world. To get the maximum profit from a unit area farmers are reporting to higher stocking densities and artificial fertilization of the ponds and supplementary feeding with good management practices they were using artificial feeds. In these culture systems with the over intensification there is a chance of stress to the growing organism. During stress pathogens present in the pond enter and cause diseases to the culture organism. The disease intensification in shrimp results in severe mortality due to stress. During the last few years’ Asian countries were severely affected with many viral diseases, particularly India faced massive economic loss in recent years due to White Spot Disease (WSD). Continuous outbreak of WSSV to tiger shrimp Peneaus monodon has spread and caused large scale mortalities and severe damage to shrimp aquaculture industry.

In a successful shrimp farming practice stocking density is one of the important and basic parameter to avoid stress. It plays a vital role in the confined environments. It addresses both the carrying capacity of the holding environment and the spatial and behavioral needs of the species. Commonly it was defined as the weight of fish/shrimp per unit volume in unit time of water flow in the holding environment (Ellis, 2001). Normally low stocking leads to economic loss and high densities affect the shrimp welfare. Sometimes higher stocking density and poor water quality management are the major reasons for many disease outbreaks.

Generally Survival, growth and production of a shrimp depends on the type of culture system (e.g. extensive, intensive and semi- intensive). Stocking rates for high-density aquaculture are typically thousand fold greater than wild environments. Understanding the relationship among density, mean size, survival and yields of semi-intensive cultured shrimp is more important to increase production efficiencies and lower the nutrient effluents. High stocking density of fish or crustaceans in ponds usually exacerbates problems with water quality and sediment deterioration. The required water quality is determined by the specific organism to be cultured and has many components that are interwoven. Growth and survival, which together determine the ultimate yield,

40

are influenced by a number of ecological parameters and management practices.

Water quality management became the limiting factor because of higher

feeding rates and greater stocking densities in the intensive farming. The

physico-chemical factors of the pond water and quality of supplementary feed

as individual or synergistically plays important role on shrimp production. The

ecosystem and biota of the culture pond may also influence the production

performance of shrimp culture. The growth and survival of shrimps are affected

by water temperature, salinity, pH and dissolved oxygen concentration

(Subrahmanyam, 1973; Verghese et al., 1975, 1982; Liao, 1977). Many studies

have aimed to increase production of shrimps through manipulating of stocking

density, fertilization, artificial feeding, opening of new lands for culture and

combination of other species into the culture system (Verghese et al., 1975;

Chakraborti et al., 1985; Krishna, 2006).

Several authors described about the growth in shrimp culture systems

based on stocking density. (Cailout et al., 1976; Sedgwick 1979; Maguire and

Leedow 1983). Some authors have reported an inverse relationship between

growth and stocking density (Lee et al., 1986; Sandifer et al., 1987; Whay-

Ming and Yew-Hu, 1992; Daniels et al., 1995). Through many researchers

stocking densities have been established for several species of penaeid shrimps

in high-salinity systems, including P.monodon (Ray and Chien 1992),

P.vannamei (Wyban et al., 1987; Sandifer et al., 1987), P.penicillatus (Liao

and Chien 1990) and P.semisulcatus (Al-ameer and Cruz 2006).

No proper research has yet been done on the effect of stocking density in

long term survival and growth performance of L.vannamei. Hence it was aimed

to evaluate the effect of different stocking density on the survival and growth

of L.vannamei for the present study.