3.0. ARTIFICIAL FEED FORMULATION FOR SUB …shodhganga.inflibnet.ac.in/bitstream/10603/61785/9/09_chapter 3.pdf · ARTIFICIAL FEED FORMULATION FOR SUB ADULTS OF SPNY LOBSTER PANULIRUS

3.0. ARTIFICIAL FEED FORMULATION FOR SUB ADULTS OF SPNY

LOBSTER PANULIRUS HOMARUS

3.1. INTRODUCTION

On a global scale, aquaculture accounts approximately 26% of all fish harvests

and aquaculture represents 33 % of all “food fish” excluding clupeiformes harvest (FAO,

2001). In global fish supply most of the growth is derived from aquaculture. Aquaculture

is where future growth will come from. It is the focus of pivotal policy decisions

regarding ownerships and management in aquatic environment and will dominate the

international trade and marketing of many species, especially high value species and

competition from aquaculture is an important catalyst for change in fisheries

management(Anderson,2002).

The contribution of aquaculture to global supplies of fish, crustaceans, mollusks

and other aquatic animals continue to grow, increasing from 3.9% of total production by

weight in 1970 to 27.1% in 2000 and 32.4% in 2004. Aquaculture continues to grow

more rapidly than all other animal food producing sectors. World - wide, the sector has

grown at an average rate of 8.8% per year since 1970, compared with only 1.2% of

capture fisheries and 2.8% for terrestrial farmed meat production systems over the same

period. Production from aquaculture has greatly outpaced population growth, with per

capita supply from aquaculture increasing from 0.7kg in 1970 to 7.1kg in 2004,

representing an average annual growth rate of 7.1%. From a production of below 1

million tons in the early 1950s, production in 2004 was reported to have risen to 59.4

million tones, with a value of US $ 70.3 billion. This represents an average annual

increase of 6.9% in quantity and 7.7% in value over 2002. In 2004, countries in Asia and

Pacific region accounted 91.5% of the production in quantity and 80.5% of the value

(FAO,2006).

Spiny lobsters are world’s one of the most valuable sea foods with high market

appeal in Asia, Europe and America. Aquaculture appears to be the only long term way

of meeting market demand for spiny lobsters. Laboratory scale rearing from egg to

puerulus larvae has been achieved for many species of spiny lobsters including Panulirus

japonicas, Panulirus longipes, Jasus lalandii, Jasus edwardsii and Sagmariasus

verreauxi (Kittaka,2000, Matsuda and Yamakawa, 2000). However, commercially viable

hatchery production of the spiny lobster is still some time away, although the recent large

scale larval rearing of the tropical species P. ornatus, feeding dry pelleted feed in a

commercial shrimp hatchery in Australia ( Smith et al., 2003). Till hatchery production

becomes commercially viable, the only practical way of increasing the volume of

marketed spiny lobster is to capture juveniles from the wild and on grow them to market

size, thereby circumventing the high natural mortality that otherwise occurs (Philips et

al.,2003).

One of the main reason attributed for the non-implementation of large scale

lobster culture practices throughout the world is shortage of seed. Presently lobster

culture industry is mainly focused on natural seed resource which is too inadequate to

meet the fast growing demands. Though, it has been possible to successfully complete the

larval development of some species in the laboratory by countries like Japan, till now a

breakthrough has not been achieved. Spiny lobster aquaculture is developing rapidly as

an important industry . Lobster culture practiced in countries like Japan, Taiwan,

Vietnam etc are by stocking natural seeds .In Japan, extensive culture of Panulirus

japonicus is being practiced along the southern coast. In Taiwan, a well organized

industry for the culture of Panulirus homarus is developed by stocking small sized

lobsters obtained from the wild and grown in ponds for 16 months time period till they

attain marketable size. The lobsters are also grown in farms containing 5 to 6 grow out

ponds having 1.3 hectares of water producing about 1,50,000 marketable size lobsters on

annual basis (Sakthivel,1994). In Vietnam, cage culture of lobsters are being practiced

since 1992 with significant expansion from the year 2000 for four species such as

P.homarus on a major account followed by P.longipes, P.ornatus and P.stimpsoni on a

lesser magnitude. The culture is done by stocking wild seeds using three types of cages

such as floating, wooden fixed and submerged which are operated within 10-20 m depth.

The approximate production of lobsters from 35,000 grow out cages was 1,500t. (Tuan

and Mao,2004). Throughout the culture period, the lobsters are fed exclusively with fresh

,whole or chopped fish and shell fish. In New Zealand focus on sea based cage culture

of lobsters from commercial scale on growing of pueruli of Jasus edwardsii in land based

facilities was initiated during 2001.( Jeffs et al., 2000; Jeffs, 2003). So far very little

research has been conducted for the development of artificial feed for spiny lobsters

throughout the world.

For the past two decades, India has made tremendous progress in the aquaculture

sector, producing significant quantities of food, employment and income. Marine

shrimps, bivalves, fin fish including fresh water fish and prawns were the major resources

significantly contributing in the production .However, lobster culture remains one of the

areas which have not been given proper attention for the development and expansion.

Requirement of considerable travails and associated difficulties in arriving standardized

technology for the larval rearing especially on nutritional aspects of different life stages

of the lobsters, prolonged larval life cycle including longer life span, limited species

diversity are the major reasons attributed for this scenario (Mitchell, 1971; Dexter, 1972;

Kittaka and Eishi, 1988).One of the main limitations for lobster culture has been high

production costs and lack of high quality formulated feed (Asbijorn, 2005). Success of

intensive commercial culture of lobster depends heavily on the availability of a relatively

inexpensive feed (Abramo et al., 1981).

Focus on commercial culture or farming of lobsters would offer one of the

effective management strategies to sustain the fishery also by reducing fishing pressure

on the wild stock. Experimental scale culture of spiny lobsters has showed their

hardiness and high growth rate (Radhakrishnan and Vijayakumaran,1984a,b,

Srikrishnadhas et al.,1983),little aggression and cannibalism even under high stocking

density (Van Olst et al.,1980).The method of rearing naturally available puerulus and

juveniles was suggested (Ingle and Witham.,1968; Chittleborough.,1974 a,b and Serfling

and Ford.,1975;James., 2007).

At present in India, lobster culture is practiced on a small scale basis by

fattening few species such as Panulirus polyphagus and Panulirus homarus along few

places in three states such as Bhavanagar in Gujarat, Kanyakumari District of Tamil

Nadu and Vizhinjam in Thiruvananthapuram District of Kerala. Growth response

studies of P.homarus under laboratory conditions were studied (Srikrishnadhas et

al.,1983,Radhakrishnan and Devarajan,1986 Kaleemur et al.,1997).An illustrated

account on cultivable species of Indian lobsters, their distribution, biology including

prospects for lobster culture using naturally available baby lobsters by cages and trays

were described (Suseelan et al.,1992). Recent experimental scale culture of Panulirus

homarus and P.polyphagus conducted respectively at Vizhinjam in Kerala , Kanyakumari

in Tamil Nadu and Veraval in Gujarat proved that small sized lobsters can be

successfully grown to marketable size within 3 months in cages fed with fresh/live feeds

such as mussels and trash fishes (Syda Rao et al., 2010, Lipton et al.,2010 and Gulshad

Mohammed et al., 2010 ).

In India, aquaculture industry is mostly dependent on commercial feeds mainly

imported from countries like Japan, China, Taiwan etc. Various Research Institutes like

CIBA, CMFRI and CIFA has developed feeds for many of the ornamental fishes and

cultivable species of shrimps such as Penaeus monodon, Fenneropenaeus indicus etc.

However, most of the feed formulations remains on experimental basis or are used

mainly by a limited scale. In India so far a standard diet has not been perfected for spiny

lobsters. Growth aspects of lobsters fed with artificial feed are very few except for the

recent studies for spiny lobsters (Ayyappn, 2002, Lamek et al ., 2004) and for mud

lobsters (Joe Kizhakudan, 2004 and 2011). Most of the studies on spiny lobsters were

related to the growth aspects of P.homrus grown under confinement by feeding

natural/live feeds (Radhakrishnan,1996; Nair et al., 1981; Radhakrishnan et al., 1998;

Kaleemur Rahman et al.,1997; Bindhu Varghese,2008). Success has been reported from

some parts of the world such as USA, Tasmnia, Australia ,Argentina and Scotland in

developing formulated feed for adults of few species. In view of feed being the major

operational expenditure accounting 30-70 % of aquaculture system, development of cost

effective ,eco-friendly and nutritionally well balanced suitable feed would be considered

as one of the thrust area to be given due attention (Conklin et al.,1989, Paul Raj,1997,

Crossland,1988, Jeffs and Hooker, 2000).

In any aquaculture system, operational cost is mainly focused on the cost

of feed and net profit rely solely on the production/ growth of the animal. This in turn

will depend upon the quantity and quality of the feed consumed by the animal,

irrespective of favorable environmental conditions. Food plays a major role in the

growth of the animals by supplying energy and nutrients in addition to pampering taste.

It is essential for overall health maintenance of the animal. It is also known that both

energy and nutrients are required in specific amount and any imbalance whether

deficiency or excess would adversely affects the health of the animal. Effective feed

formulations are those which contain adequate levels and balanced proportions of

essential nutrients and additives optimally processed and produced in a form easily

acceptable and palatable for the species under culture (Paul Raj,1997). Formulation of a

good feed depends upon a careful evaluation and selection of its ingredients. An

inferior quality feed when provided, will have a negative impact on survival, growth and

overall production from the culture system leading to monetary loss to the farmer. In the

present study an attempt was made to formulate artificial diets for sub-adults of

P.homarus by preparing a series of test diets and their impact on growth performance

was assessed over a period of 3 months under laboratory conditions.

3.2. MATERIALS AND METHODS

For the present studies, rock/spiny lobster Panulirus homarus was selected as the

experimental animal. It is a decapod crustacean belonging to the family palinuridae,

naturally seen in marine system and is endemic to peninsular India and abroad. Holthuis

(1964) proposed synonymising Panulirus dasypus and Panulirus burgeri as Panulirus

homarus and this was accepted by later authors (Gordon,1953; George,1963 and Kubo,

1963). Taxonomic position of Panulirus .homarus is given below. (FAO, 1984)

Classification

Phylum : Arthropda

Class : Crustacea

Order : Decapoda

Suborder: acrura Reptantia

Family : Palinuridae

Genus : Panulirus

Species : homarus

3.2.1. Description of the experimental animal

It is a medium sized lobster growing to a maximum of 30 cm in body length. The

lobsters are called as spiny lobsters because of the presence of numerous spines on their

body. The characteristics colors of the lobsters are due to carotenoids, largely by the

asetaxanthin present in pigmented layer just beneath the epicuticle of the exoskeleton.

They have very good chemoreceptive ability and are thereby known to discriminate

among odours, in addition to the obvious and well developed senses that allowed

lobsters to perceive visually and chemically. Lobsters also find out a great deal about

their surrounding environment through the sense of touch. They are heterosexual, strictly

marine throughout their life. After attaining maturity at the age of two to two and half

years, they reproduce in the inshore waters. Females carry eggs from I to II months

cemented to the pleopods under the tail. The incubation time of the eggs are highly

variable and temperature dependent. Fertilized eggs after completing embryonic

development in the berry of the mother lobsters, hatches out as a flat transparent pelagic

larvae called phyllosoma. The body of the phyllosoma larvae is extremely dorsoventrally

flattened, leaf like, transparent and with highly setose appendages. The larval stages of

the spiny lobsters last much longer and are thus much more liable to natural mortality,

including predation. The complex larval stage of phyllosoma lasts for a long period of

3 to 22 months and it gets transformed to puerulus stage. The puerulus soon after settles

down for benthic life and they transforms in to a juvenile baby lobster. The puerulus of

palinurides is approximately 8-10 mm in carapace length. During these period, drastic

changes in their behavior occurs. The puerulus stage generally extends to a duration of 2-

3 months. It subsequently settles and molts in to juvenile stage exhibiting same

morphology of adults.

Lobsters are primarily scavengers. They forage food at low intensities of light.

Their food consists mainly of crustaceans, polychaetes, mollusks and gastropods. Under

captivity, cannibalism is observed especially at the time of molting when they attack

newly molted animals. Lobsters exhibits autonomy and have the power of regenerating

lost body parts. The regeneration off lost body parts take place during the next molting

and the time required for its completion depends on the time intervals between the

completion of molting.

3.2.2. Lobster collection, packing ,transportation and maintenance

Twenty four numbers of sub adults of spiny lobster P.homarus below 100 g body

weight were bought from fishermen operating traps at Kadiapatanam fishing center,

Kanyakumari district of Tamil Nadu along the south east coast of India. The lobsters

were selected based on their external appearances with all appendages, intact exoskeleton

and good pigmentation. Lobsters were transported to the laboratory, Centre for marine

Science and Technology, Rajakkamangalam under moist conditions. Prior to packaging,

lobsters were dipped for five minutes in 13 0 C cooled sea water to lower their biological

activity. The lobsters were then kept inside a rectangular shaped polystyrene box by

placing over a layer of news paper sprayed lightly with 130 C cooled sea water. The

lobsters were then wrapped individually in suitable size of news paper and placed serially

inside the box. They were placed one upon the other by placing water sprayed news paper

like a sandwich. Inside the corners of box, 2 plastic bottles filled with 130C cooled sea

water was also kept to maintain ambient temperature. Finally a thin layer of news paper

was again spread on the top of the lobsters and the box was tightly closed .Lobsters in

this state will be inactive and can be transported without any mortality even for long

hours ,the usual practice followed by entrepreneurs for export of lobsters from India.

Lobsters exposed to sun and wind will suffer serious drying effect which weakens

them. In order to prevent this, lobsters needs to be packed with care, preferably in single

layers separated by damp sacking, which helps to maintain a moist atmosphere. The

moist air is essential, as it prevents the gills from drying out and enables respiration to

continue. Lobsters packed and transported in this method can survive out of water for

quite long periods (Beard and Gregor ,1991).The lobsters were then acclimatized to

ambient laboratory condition in a 300 liter capacity fiber glass tanks using freshly

collected filtered sea water provided with continuous aeration.

3.2.3. Water quality maintenance

Every day morning about 85% of water from the experimental tanks was

siphoned out along with left over feed and fecal matter. Continuous aeration was

provided so as to maintain oxygen level at desired level. In the present study, the

experimental tanks were maintained during the entire experimental period following good

water quality practices with utmost care, without causing stress to the lobsters. Since

quality of the feed significantly affects water quality factors such as ammonia excretion

including oxygen consumption , parameters such as salinity, dissolved oxygen, pH,

temperature and ammonia were monitored every alternate day.

a.Water temperature

The water temperature in the experimental tanks was recorded using thermometer

at early morning, mid-noon and evening.

b.Salinity

The salinity of the experimental tanks was recorded using refractometer at each

time during the collection , storage and also during the experimental period.

c. Hydrogen ion concentration (pH)

The pH of water samples was recorded using digital pH meter once in a week.

Dissolved oxygen (Winkler’s method) (APHA, 1985)

Dissolved oxygen from each experimental tank was recorded once in a week.

Principle

Manganous chloride was added to the water samples drawn from experimental

tanks to form manganous hydroxide. In the presence of dissolved oxygen in the water

samples, the manganous hydroxide gets oxidized to mangenic hydroxide. Addition of

alkaline iodide along with concentrated H2SO4 liberates equal amount of iodine as that of

oxygen which was used for the conversion of manganous hydroxide into mangenic

hydroxide. Hence the amount of iodine liberated is measured as the indication of

dissolved oxygen in the sample. The liberated iodine is then estimated by titrating against

0.025 N sodium thiosulphide using 1% starch solution as indicator.

3.2.4. Reagents

a. Manganous Chloride

Manganous chloride solution was prepared by dissolving 4g manganous chloride

in 100ml distilled water.

b. Alkaline iodide

The reagent was prepared by dissolving 50 g sodium hydroxide in 1 liter distilled

water followed by adding 16.6 g potassium iodide, shaked well and cooled to room

temperature before use.

c. Sodium thiosulphate (0.025 N)

This reagent was prepared by adding 6.750 g of sodium thiosulphate dissolving

in 1 liter distilled water.

d. Starch solution (1%)

The indicator solution was prepared by dissolving 1 g starch in 100 ml distilled

water and cooled to room temperature.

Procedure

The water sample was filled in a clean reagent bottle without entangling air

bubbles. To this sample, 2 ml manganous chloride and 2 ml alkaline iodide were added.

The precipitate then formed was allowed to settle for some time. Then 2 ml concentrated

H2SO4 was added to dissolve the precipitate. From this, 50 ml of sample was taken in a

conical flask as an indicator. This was titrated against 0.025 N sodium thiosulphate from

the burette. Disappearance of blue color was noted as end point. The titration repeated for

each sample to get concordant values. The dissolved oxygen present in the sample was

calculated using the following formula.

Calculation

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M = Molarity of sodium thiosulphate

E = Equivalent weight of oxygen

1000 = To express dissolved oxygen per liter

0.698 = To convert parts per million to ml of O2 per liter.

VT = Volume of titrant

Vs =Volume of the sample

Ammonia

Phenol-hypochlorite method (Soloranzo, 1969)

The principle involved is the formation of a blue colored indo phenol due to the

reaction between phenol-hypochlorite and ammonia. The intensity of blue color was read

at 640 nm in a spectrophotometer.

Reagents

Alcoholic phenol

Alcoholic phenol solution was prepared by dissolving 5 g phenol in 50 ml

ethanol.

Sodium nitroprusside

Sodium nitroprusside solution was prepared by dissolving 0.5 g in 100 ml

distilled water and kept in dark bottle.

Sodium citrate

This solution was prepared by dissolving 20 g trisodium citrate and 1 g

NaOH in 100 ml distilled water.

Oxidant

Oxidant was prepared by mixing 20 ml sodium citrate in 5 ml sodium

hypochlorite solution.

Procedure

To a known volume of sample (5 ml), 0.2 ml sodium nitroprusside, 0.2 ml

alcoholic phenol and 0.5 ml oxidant were added. The intensity of color developed was

then read in a spectrophotometer at 640 nm.

Blank

Blank was prepared by adding 0.2 ml sodium nitroprusside, 0.2 ml alcoholic

phenol and 0.5 ml oxidant also adding 5 ml distilled water.

Standard solution

The solution was prepared by dissolving 3.182 g NH4CL in 1 liter distilled water.

3.2.5. Ingredients selection

The ingredients for the formulation of dry compounded artificial feed for sub

adults of P.homarus was selected based on their nutrient status, animal preference

suitability in terms of form, structure, texture, flavor, low price, attractiveness, keeping

quality and local availability in terms of steady supply. The ingredients selected with

their characteristic properties are given below.

Fish meal

The fish meal was prepared from dried anchovies procured from commercial fish

market .Fish meal is the mostly preferred single most ingredient used in aquaculture

diets .It is a highly concentrated nutritious feed supplement consisting high quality

proteins, minerals including vitamins of B group . They also have excellent flavor and

associated good texture. Fish meal is a preferred ingredient in balanced rations for

shrimp because of its high protein content, essential amino acid composition particularly

lysine and methionine, fatty acids, mineral content with acceptable palatability and

digestibility. Anchovies are available though out the maritime states of India in

considerably large quantities. Though smaller in size, it is an important sea food item

mostly consumed by the coastal communities. The protein and carbohydrate contents

estimated in the laboratory was recorded as 58.5% and 1.47 % respectively.

Mussel meal

Perna indica ,commonly known as brown mussel collected from Khadiapatanam

centre was brought to the laboratory, boiled, dried, powdered and used .They have

excellent flavor and appealing golden yellow color when dried. Brown mussels forms

one of the most favored food items of the experimental animal .Brown mussels are also

belongs to the one of the favored sea food items preferred by the coastal communities.

Dishes made of mussels are also found to be an important delicacy served in restaurants

due to their extrinsic qualities .Mussels are available abundantly along the east and west

coast of India throughout the year. The protein and carbohydrate in the mussel meal

estimated in the laboratory was 76.8 and 2.4 % respectively.

Hippa meal

Hippa hippa was collected from Rajakkamangalam inshore waters and dried in

hot air oven ,powderd and used . It is an important sources of marine protein .They have

comparatively strong flavor and lobsters are particularly attracted to this quality. They are

crisp especially when dried. They are also consumed by traditional fishermen in some

parts of the coastal areas along Tamil Nadu and Kerala. They are distributed in sandy

near shore waters along south west coast of Kerala and Tamil Nadu. The protein and

carbohydrate content estimated in the laboratory was 35 % and 1.73% respectively.

Soya bean meal

Soya bean meal procured from commercial market was used. It is an important

source of nutritious food ingredient characterized for their high quality protein, low

saturated fat with richness of vitamins especially B-complex and also minerals such as

magnesium, calcium, iron, potassium, copper including fiber. The protein and

carbohydrate content was estimated in the laboratory was 43.4 % and 2.19 %

respectively.

Ground nut oil cake

Ground nut oil cake was procured from local commercial market , powdered

and used . It is one of the important and cheap by-products obtained from ground nuts .

They have substantial quantities of protein, carbohydrate and fat , including fiber. The

crude protein and carbohydrate was estimated in the laboratory was 62 % and 15.2%

respectively.

Casein

Casein was procured from local commercial market. It is an important

protein source derived from the milk. The protein and carbohydrate content was

estimated as 78.6 % and 1.3 % respectively.

Di-sodium hydrogen orthophosphate

Di- sodium hydrogen orthophosphate was purchased from commercial market. It

is one of the important sources of minerals available in powder form which is very much

essential for the health of the animal.

Sea weed (Sargassum whitii)

Sargassum whitii an important sea weed available in plenty along the Tamil

Nadu coast was collected from Kanyakumari inshore waters was brought to the

laboratory,cleaned,sun dried , powdered and used .Seaweeds are rich in proteins,

vitamin, minerals and trace elements. The protein and carbohydrate content estimated in

the laboratory was formed as 25.8 and 2.36 % respectively .Sea weeds are used as food,

fodder, fertilizer as well as source for drugs and therapeutically important substances. In

India about 720 species of sea weeds are available of which, 60 are commercially

important. They occur abundantly along the coast of Tamil Nadu, Gujarat, Lakshadweep

and Andaman Nicobr Islands .Use of sea weed meals as a binder in fish and prawn feed

increase physicochemical standards thereby maintain water quality. (Kaladhran et

al.,1998).

Cod liver oil

Cod liver oil purchased from local commercial market was used. It is a very stable

and rich source of lipid, having very good binding properties. Fish oils also improve feed

palatability.

Vitamins and minerals

Vitamins are chemically diverse group of organic substances critical for the

maintenance of normal metabolic and physiological functions. Minerals are important

constituents of the structural components of tissues and shells . Many of the minerals

serves as components of enzymes, vitamins, hormones, pigments and as a co-factor in

metabolism or as enzyme activators. In the present study “Supradin” a multi vitamin and

mineral tablet available in commercial market were used . The composition is given

(Table.3.2)

Binders

A total of eight binders sourced from plants such as tapioca powder, agar agar,

gum arabic, wheat flour, stick on ,guar gum including synthetic binders such as gelatin

and sodium alginate were used for the formulation of experimental diets for P.homarus.

Major qualities of the binders are given .

Agar agar

Agar agar was purchased from commercial market. It is one of the important

source of marine protein mainly used as culture media for Microbiological studies. It is

also used for culinary, ice cream and in tooth paste making industries as a major

ingredient. In food technology, agar is used for gelling (Kaladharan 1998). The protein

and carbohydrate content estimated in the laboratory was 9.8 and 3.62% respectively.

Tapioca powder

Tapioca powder was purchased from local commercial market . It is an

important source of binder extensively used in commercial feed preparation of aquatic

animals. Tapioca is also known for one of the major sources of carbohydrate and also

contains fiber and low amount of fats. It is cheap and available in plenty along south

India. It is mainly used for human consumption. The protein and carbohydrate content

estimated in the laboratory was 2.2 and 71 % respectively.

Stick on

Stick on is an important stable phytochemical available in liquid form . It was

procured from C.P. Aquaculture Pvt Ltd from Chennai. It blends easily with firm grip

with feed ingredients thereby preventing the dispersion of feed particles in water by

maintaining their bio-availability. It is safe, eco -friendly and also does not cause any

deterioration of water quality. It contains natural proteins based feed attractants .

Wheat flour

Wheat flour was purchased from local commercial market. It is an

excellent source of complex carbohydrates known as polysaccharides. It forms visco-

elastic dough when mixed with water and the presence of water compatible gluten protein

allows the dough to swell and interact with the ingredients more quickly.

Guar gum

Guar gum was procured from local commercial market. It is an important source

of emulsifier derived from guar beans available as free flowing fine powder. It is one of

the most efficient water thickening and soluble fiber that act as a laxative composed of

sugars, galactose and mannose.

Sodium alginate

Sodium alginate was procured from commercial market. It is a chemical

compound or gum extracted from the cell walls of brown algae. As a stabilizer,

thickening and emulsifier, it is widely used in the food industry.

Gum arabic

Gum arabic was procured from local commercial market. It is a natural gum made

of hardened sap taken from acacia tree. It is a complex mixture of polysaccharides and

glycoprotein used primarily in the food industry as a stabilizer and a binder.

Gelatin

Gelatin was procured from local commercial market. It is the most common

protein in the animal kingdom obtained by the thermal denaturizing of collagen derived

from the skin, white connective tissue and bones of domesticated animals. It is

translucent, colourless, odourless, brittle,tasteless , solid substance which is commonly

used as a gelling agent in food industries. It forms a solution of high viscosity in water

which sets to a gel on cooling.

3.2.6. Basal feed formulation

The primary objective of feed formulation is to provide an acceptable diet having

good nutritional status for the candidate species so as to get better yield at minimum

expenditure of money, effort and time .The ingredients were selected based on the

proximate composition and basal feeds were prepared.

The ingredients were selected after nutrient evaluation based on protein and

carbohydrate contents. Selected ingredients were hygienically sun-dried for five hours

and subsequently dried in hot air oven maintained at 550 C. In this study an attempt was

made to formulate artificial dry compounded feed for sub adults of P.homarus. The

ultimate successes of aquaculture rely on good quality feed and water. The experimental

studies were carried out by preparing a series of test diets using natural resources of plant

and marine animals, mostly cheap, readily available and also having good keeping

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Plate: 5. Basal Feed

qualities with substantial nutrient status . Some of the main ingredients used were

also preferred by the animals as food in their natural habitat.

The ingredients were made in to fine powder using electrical mixer and were then

quantified and homogenized well and made in to dough using distilled water. The dough

was then steam cooked for 10 minutes, subsequently cooled and added with casein.

Estimated quantities of vitamin/mineral mix were also added to the thoroughly

homogenized dough and made in to round pellets of approximately 4 mm diameter

without adding binders. The pellets were then sun dried for one hour subsequently kept in

hot air oven maintained at 550 C till the moisture content of the pellets were reduced to

minimum of 7 %. The basal diet so prepared was stored well in air-tight containers for

further studies. The proximate composition of basal feed was estimated as protein 54.5 %

, carbohydrate17.6 % .

a.Experimental trial on P.homarus for feed acceptance

The lobsters were carefully released to tanks filled with filtered sea water

provided with aeration. After an acclimatization period of 12 hours, the basal feed were

provided to the lobsters .The basal feed was found palatable as recorded from their

immediate consumption, soon after the feed was introduced in the experimental tanks.

However, stability of the basal diet was found as very low.

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b. Binder selection

In order to improve the stability of the basal diet, 35 test diets were prepared

through series of experiments by incorporating different concentration and combinations

of binders in the basal diet so as to develop cost-effective, stable, well appealing diets

having substantial nutritional qualities that will enhance ideal growth for lobsters under

controlled conditions .

3.2.7. Feed stability

Observations were made in the laboratory to test water stability/endurance of the

diets for varying exposure periods in the sea water by standard methods such as dip,

string and pouch methods ( Ahamad Ali et al., 2006). In the first two methods,

percentage of nutrient leaching or dry matter leaching was recorded as an index of water

stability.

(i) Dip/immersion method

The stability of the feed was estimated as the percentage of nutrient leaching as an

index of feed stability by sea water immersion method. Experimental diets in the form of

round shaped pellets were placed in plastic tanks containing 75 liters of filtered sea water

provided with continuous aeration for different time intervals such as 2,4,6and 8 hours.

The pellets were taken out from the tanks after the stipulated time and washed in fresh

water to remove traces of sodium chloride adherent on and water content was removed

slightly by using tissue paper and kept for drying in hot air oven maintained at 600C .

Weight of the pellets was recorded before and after sea water immersion followed by

drying .

(ii) String method

The stability of feed was estimated as the feed detachment from the string as an

index. Few pellets with uniform length were taken and recorded their initial weight. Each

pellet was tide at their centre separately using strings and slowly suspended in one liter

beaker filled with distilled water. The suspension of pellets was adjusted in such a way

that it was just below the water surface. The times taken to fall each pellets from the

string were recorded separately. The mean value of time was used for the estimation of

pellet stability in water .Higher the time, better the stability of feed.

(iii) Estimation of stability using pouch method

Cone shaped pouches made of 1 mm nylon mesh were washed and dried at 60 0 C

to constant weight in an air oven. Known quantity of uniform sized pellets were kept in 4

pouches introduced in 4 petri- dishes placed at different points in the bottom of the tank

filled with sea water provided with aeration. At different intervals of time each pouches

with the pellets were taken from the tank and examined for their physical shape. The

pellets were then dipped in fresh water for 5 minutes to remove the salt content

.Subsequently the pouches with the pellets was dried at 70 0 C till they attained constant

weight .The difference in the initial weight and final weight of the pellets i.e., before and

after sea water immersion was recorded as nutrient loss for set time intervals .The

percentage loss of dry matter was estimated by formula used in immersion method. The

results were recorded as mean values obtained for dip, string and pouch methods.

3.2.8.Effects of binders on the pellet stability and palatability of formulated dry

compounded diets

A total of eight binders sourced from Plant and synthetic origin were selected for

the preparation of a palatable and stable diet for the sub adults of Panulirus homarus. The

binders used were gelatin, sodium alginate, tapioca flour, wheat flour, stick on, agar agar,

gum arabic and guar gum.

3.2.9.Experimental trial on P.homarus for feed acceptance and stability

A total of 35 combinations and with different concentrations of binders were

added in basal diets and 35 types of dry compounded feeds were prepared and tested their

stability and acceptance by experimental lobsters .The lobsters were carefully released in

to 100 l capacity tanks filled with filtered sea water provided with aeration. After an

acclimatization period of 12 hours, experimental diets were provided to the lobsters. The

lobsters were then monitored to record feed acceptance as well as rejection. The accepted

feed was then recorded as palatable. Observations were also made to test the water

stability of the diets for varying exposure periods in the sea water by standard methods

.The stability of the diets was carried out by testing the endurance of the diets in sea

water for longer periods and the palatability was determined based on the diet acceptance

in terms of consumption by lobsters .The percentage of dry matter leaching was recorded

as an index of water stability .The stability of different feed was tested at specific

intervals of 2 hrs,4 hrs, 6 hrs to a maximum of 8 hours. Different stability grades were

given to pellets such as mild representing 55% to 59 % pellet stability , moderate 60 to

79 % and good ranging from 80 to 89% based on 8 hrs sea water immersion.

The percentage of feed leaching was estimated using the formula given below.

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Experimental diet preparation

Among 35 combinations of binders , 3 combinations of binders were identified

for their better performance based on feed acceptance and stability when incorporated

in the feed . The selected combinations of binders were then used for the preparation of

6 types of dry compounded feeds using different ingredients so as to derive a well

appealing diet based on the growth performance of lobsters which was estimated by

conducting 3 months experimental culture of lobsters feeding all the six formulated

feeds.

Experiments on Growth performance

The experimental set- up is shown (Plate 7) . For diet evaluation and growth

performance studies a total of 24 numbers of sub-adults of P.homarus belonging 100-

118 mm sizes were procured from fishermen operating traps at Khadiapatanam, carefully

transported and acclimatized for 24 hours in the laboratory. Morphometric details of all

the lobsters were recorded before the start of the experiments as per FAO standard

methods. For the studies two lobsters of P.homarus i.e., one male and one female of

almost same sizes were stocked in 100 liter capacity ,serially interconnected rectangular

plastic tanks. A 0.1 H.P motor was used to pump fresh sea water stored in 750 liter

capacity sump. Daily sea water was pumped to the over head tank provided with

cartridge filter and the filtered sea water was allowed to flow to each tank through the

interconnected PVC pipes .In each tank ,two numbers of hollow PVC pipe pieces

measuring 18 cm length and 9 cm diameter were placed so as to mimic a hide out for the

lobsters . Aeration was provided continuously in all the tanks throughout the

experimental period. The experimental set up was maintained almost in dark condition as

the color of the tanks were deep blue and all the tanks were further enclosed with a

similar colored nylon curtain.

Since lobsters are mainly nocturnal feeders, feeding was done as a single dose

daily at 6 PM throughout the experimental period. Every day morning left over feed

along with fecal strands were siphoned out from each tank followed by 85 % water

replacement .The replaced water from each tank were filtered through a 50 liter capacity

biological filter connected to the sea water sump and was mixed with fresh sea water

at 1:1 ratio and were re-used in the system there by maintained minimum sea water

usage . Following this method, water is filtered twice before using in experimental tanks

assuring very good water quality. Daily left over feed and fecal strands were separated at

the time of water exchange and the unfed pellets thus collected were rinsed in fresh water

to remove traces of salt and finally stored in stainless steel dishes and dried at 550C over

night in hot air oven. The dried left over feed was weighed in electronic weighing balance

having one milligram accuracy and the difference between initial and final feed quantity

was recorded to estimate the amount of feed consumed by the lobsters.

Details on various aspects of growth expressed in terms of growth per day, food

consumption, feed conversion ratio, specific growth rate etc of Phomarus grown by

feeding 6 types of formulated diets were estimated using the following formulas.

Production = Final wet weight of the animal-Initial wet weight of the animal(g)

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Moulting

In the present study, details on moulting aspects of P.homarus fed with 6 types of

formulated experimental feeds were recorded separately for males and females. Moulting

rate, moult day-1 and moult cycle were estimated using the following formulas.

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Biochemical analysis

In order to find out variations in biochemical changes in selected tissues of

lobsters such as muscle, gill and hepatopancreas the tissues of acclimatized experimental

lobsters were dissected out under aseptical conditions and weighed , immediately before

the experiments and tissues of all the experimental lobsters after the 90 days feeding

experiments. The samples were then kept separately in hot air oven at 60oC for 72-96

hours to remove moisture content and were stored in desiccator till biochemical analysis.

The above tissue samples were then analyzed by standard methods to estimate protein,

carbohydrates and lipid contents of the control and experimental animals fed with 6 types

of formulated diets.

Estimation of protein (Lowry et al., 1951)

Reagents

Ethanol (80%)

By diluting 80 ml of ethanol in 20 ml distilled water, 80 % of ethanol prepared.

NaOH (0.1N)

By dissolving 400 mg NaOH in 100 ml distilled water,0.1 N sodium hydroxide

solution was prepared.

NaOH (1N)

For the preparation of 1N NaOH, 4 g of NaOH was dissolved in 100 ml distilled

water.

Solution A

Solution A was prepared by dissolving 2g sodium carbonate in 100 ml 0.1N

NaOH.

Solution B

Solution B was prepared by dissolving 500 mg copper sulphate in 1% sodium

potassium nitrate (1g sodium potassium tartarate in 100 ml distilled water).

Solution C

Solution C was prepared by mixing 50 ml solution A with 1 ml solution B.

Folin phenol reagent

Folin phenol reagent was prepared by mixing 1 ml folin phenol with 1 ml

distilled water.

Blank

For blank 0.5 ml 1N NaOH, 5 ml Solution C and 0.5 ml folin phenol were added.

Procedure

A known amount of sample was taken and ground well with 80% ethanol. It

was then centrifuged for 15 minutes at 5000 rpm .The precipitate, dissolved in 1 N NaOH

was made up to 5 ml. From this 0.5 ml is taken and 5 ml of the solution C was added and

incubated for 20 minutes at room temperature.Finally,0.5 ml Folin phenol reagent was

added and the intensity of color developed was read at 640 nm in a spectrophotometer.

Calculation

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Estimation of carbohydrate(Roe,1964)

Reagents

Ethanol (80%)

Prepared by mixing 80 ml ethanol in 20 ml distilled water.

Anthrone

The reagent was prepared by dissolving 50 mg anthrone in 100 ml 60% H2SO4

and 1 g thio urea.

Blank

For blank 0.5 ml 80 % ethanol was used.

Procedure

A known quantity of tissue sample was grounded well with 80 % ethanol and

centrifuged for 15 minutes at 5000 rpm. Then 0.1 ml supernatant was taken I a test tube

and 5 ml anthrone reagent was added .The test tubes were then kept in a boiling water

bath for 15 minutes and subsequently kept in a dark room for 15 minutes to cool .The

color intensity developed was read as optical density in a spectrophotometer at 620 nm.

Calculation

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Estimation of lipid (Folch et al.,1957)

Principle

Quantitative determination of sulphophosphovanilin methods depends on the

reaction of lipid extracted from the sample using chloroform methanol with sulphuric

acid, phosphoric acd and vaniln to give a red complex.

Reagents

Chloroform Methanol (2:1)

The reagent was prepared by adding 800 ml of orthophosphoric acid in 200 ml

distilled water. Then 2 g vanillin was added and kept in a reagent bottle till use.

Standard

The standard was prepared by dissolving 8 mg cholesterol in 4 ml chloroform

methanol mixture at 2: 1 ratio as a blank.

Procedure

A known amount of tissue sample was homogenized in 5 ml chloroform methanol

mixture using motor and pestle . A 0.2 ml,9 % sodium chloride solution was also added

and homogenized well .It was then transferred in to a separating funnel shaken well and

allowed to settle for few hours. After incubation, the lower layer was taken and made

up to original volume by adding chloroform methanol mixture. It was then dried in a

vacuum desiccator over silica gel .It was then dissolved in 0.5 ml concentrated

H2SO4,mixed well and was kept in a boiling water bat for 10 minutes and was cooled

.From this,0.2 ml was taken and mixed with 5 ml of phospho vanillin reagent ,mixed well

and allowed to settle for half an hour. The color intensity developed was recorded at

250 mm in a spectrophotometer.

Calculation

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3.2.10.Statistical Analysis

The data derived from the present study were subjected to relevant statistical

analysis following Zar (1974).

I Mean

II Standard deviation

III Standard error

IV One way analysis of varience

V Duncan’s Multiple Range Test

I Mean

Mean is the average number of items .It is calculat

Mean = x = �x / n

II Standard deviation(SD)

Standard deviation (S) =

Where “d” refers to the deviation of each score fro

number of samples.

III .Standard error (SE)

SE =Sd/n-1

IV One-way analysis of varience

Partitioning of total variance due to different fac

Zar (1974).Values obtained for different experiment

rows. Values obtained for each column, x, x

columns squared were divided by the number of tabul

“C” was obtained.

Correlation factor ( c) = Grand sum of all x

V Duncan’s Multiple Range Test

Mean is the average number of items .It is calculated using the formula,

x / n

II Standard deviation(SD)

Where “d” refers to the deviation of each score from mean and “n” refers to total

way analysis of varience

Partitioning of total variance due to different factors was carried out according to

Zar (1974).Values obtained for different experiments were tabulated in columns and

rows. Values obtained for each column, x, x2 were calculated. Sum of X for all the

columns squared were divided by the number of tabulated values and correction factor

Correlation factor ( c) = Grand sum of all x2/n

n = total number of observation)

ed using the formula,

m mean and “n” refers to total

was carried out according to

s were tabulated in columns and

were calculated. Sum of X for all the

es and correction factor

Total sum of square (TSS) = Sum of x2 for all columns-C

Between columns SS (CSS) =Sum of all values in each column x2/n

(n=number of values in the column)

Between rows (RSS) = Sum of all values in each row x2/n

N=Number of values in each rows.

Remainder SS = Total SS- (Between column SS + Between rows SS)

Considering the degrees of freedom for each source of variance, Mean sum of

square (MS) was calculated.

V. Duncan’s multiple range test

Post hoc tests like Duncan’s multiple range test (DMRT) (Duncan, 1955) were

applied to identify significant differences between individual treatments. All statistical

analysis were conducted using SPSS for Windows and Microsoft Excel.

3.3. RESULTS

3.3.1.Basal feed

The feed was prepared using fish meal, ground nut oil cake, soya meal and casein

as main ingredients with protein and carbohydrate content of 54.5% and 17.6%

respectively. The feed was then tested for their acceptability by sub adults of P.homarus,

and for stability upon eight hours of sea water exposure. The basal feed was found

palatable as recorded from their immediate consumption, soon after the feed was

introduced in the lobster holding experimental tanks.

The composition of feed ingredients used for the formulation of basal feed with

their proximate composition/ composition of vitamins / mineral mixture is given

(Table3.1). In this study, though the feed was accepted by the experimental lobsters

stability was low forming 45 ±4.92 % for 2 hours, 38 ± 1.88 % for 4 hours, 25 ± 3.09%

for 6 hours and 18 ± 5.94 % for 8 hours. As the feed was less stable further experiments

were carried out to improve the stability by incorporating different quantities and

combinations of binders. All the diets were then tested for stability and acceptability by

the lobsters.

3.3.2. Binder selection

The details on binder combination, pellet stability and remarks on diet

performance are provided in Tables 3.3 to 3.6. Out of the 35 combinations of test diets ,

only eight diets recorded palatability with stability. Among the test diets which showed

good palatability, the diet added with the binders such as sodium alginate incorporated

@ 3 %, agar agar @ 3 % and stick on @ 1 % exhibited the highest stability of 97.36 ±

5.88% for 2 hours,92.21 ± 4.95 % for four hours, 88.01±6.69 % for six hours and 85.55±

5.94 % stability for eight hours .Likewise, the diet prepared with Agar agar (3%),

sodium alginate (1%) and stick on (1%) also contributed good stability of 84.33±3.74 %

up to 8 hours of sea water exposure. The diets added with agar agar @ 3 %, tapioca flour

@ 3 % and stick on @ 1% exhibited lowest stability of 60.55±2.84% at eight hours of

water immersion among the palatable diets.

3.3.3.Experimental compounded diets

A total of 6 types of palatable dry compounded diets were prepared using various

ingredients such as mussel meal, fish meal and hippa meal in different combinations as

major components. The composition of 6 experimental diets with their stability details

are provided in Table 3.7 with mean percentage reduction in Fig.3.1.The crude protein

and carbohydrate contents of the experimental diet varied between 41.75 % to 54.17 %

and 14.1 % to 18.63 % respectively. The diets were prepared incorporating binders such

as tapioca powder, stick on, agar agar, sodium alginates and cod liver oil in different

combinations and proportion.

3.3.4. Stability of experimental diets

Data on the stability of experimental feeds during 2,4,6 and 8 hours of sea water

exposure are provided in Table 3.8. The percentage leaching of six experimental diets

(1,2,3,4,5 and 6) during two hours of sea water exposure were 90.01±0.87%,

89.81±0.17%, 90.31±0.62%, 97.05±0.22%,90.03±0.36%, 88.0±0.24%respectively. With

the extend of exposure duration i.e., from 2 to 4, hrs the percentage leaching of

experimental diets was also increased to 87.81±0.36% , 87.53±0.36% , 83.62±0.29%,

92.02±0.19%, 77.04±0.88% and 83.02±0.18%. At 6 hrs exposure duration, the

percentage leaching of the diets was further increased to 82.83±0.09%, 80.22±0.78%,

73.06±0.57%, , 88.01±0.86% ,70.01 ±0.09% and 70.01±0.86% respectively. At the

maximum exposure duration of 8 hours, the percentage leaching of experimental diets

were 73.85 ±0.68%, 69.04±0.87%, 68.52±0.86%, 85.22±0.86%, 60.55±0.19% and

62.45±0.99% respectively in diets numbered 1,2,3,4,5 and 6.

3.3.5. Water quality analysis

Data on the water quality parameters recorded from the experimental tanks are

provided in Table 3.9. More or less an uniform temperature ranging from 27.6±0.90 to

27.66±0.94 and salinity ranging from 35.83±0.33 to 35.86±0.36 were recorded in all

experimental tanks .The dissolved oxygen content recorded was ranged from 5.39±0.13

to 6.47 ±0.51 ml /l , PH and ammonia levels were ranged from 7.93±0.07 to 8.08 ±0.

37 and from 0.004 ±0.006 to 0.007 ± 0.003 mg/ l respectively.

3.3.6. Moulting frequency

The results obtained on the influence of six experimental diets on moulting

frequency of lobsters are provided separately for males and females during the culture

period of 90 days (Table 3.10 & 3.11). The number of moult was high for lobsters fed

with experimental diet no.3. recording 3 times. An uniform moulting rate of 2 times was

observed in P.homarus fed with experimental diets1,2,4,5 and 6. The mean inter moult

period was minimum in P.homarus males fed with diet no.2 and 3 and maximum

observed for P.homarus fed with diet no.4. More or less an uniform trend was noticed

for the female lobsters reared on the same types of test diets.

3.3.7. Key Moulting statistics

Table 3.12 and 3.13 provide the data on key moulting statistics of P.homarus

fed with experimental diets during 90 days culture period. Lobsters fed with all the

experimental diets exhibited 100% survival. The moulting rate and moult day-1 was high

for diet no.3 fed lobsters ( 3 and 0.33) and it was low for those lobsters fed with diets

1,2, ,4,5 and 6. (2 and 0.022). Accordingly low moult cycle of 30.30 days was recorded

for P.homarus fed with experimental diet no.3; whereas higher moult cycle of 45.45

days was recorded for lobsters fed with diets no 1,2,4,5 and 6.

3.3.8. Growth response

Data recorded on total length and weight of males, females and total population

of P. homarus at the beginning and at the end of the experimental period of 3 months

are presented in Tables 3.14 to 317 . The result revealed although increase in total

length and weight was recorded by all the lobsters fed different diets, higher values were

obtained for diet no.3 fed lobsters than lobsters fed with remaining 5 diets . Moreover ,

this increase in length and weight between diet no.3 fed lobsters and diets 1 and 2 and 4

to 6 fed lobsters was statistically highly significant (p<0.01).

The maximum increase in total length of both males and females of P.homarus

fed with diet 3 was recorded as 6 mm with a body weight gain of 7.13±0.16 g and

minimum length increment was registered by P.homarus fed with diet no 1 recording

3.33 ± 0.51 mm increase in TL with 3.83 ± 0.33 g body weight. Fig.3.2 shows the data on

the percentage increase of these tested variables over the initial values. Table 13 provide

the data on weight increment (growth), feed consumed, food conversion ratio (FCR),

specific growth rate (SGR) feed conversion efficiency (FCE) and growth day -1 of P.

homarus fed with all the six experimental diets over three months culture period. The

maximum growth per day was recorded as 0.36 ± 0.24 g was attained by P. homarus fed

with diet no 3 and better growth per day of 0.254 ± 0.16 g was recorded for P. homarus

fed with diet no 2. In the remaining lobsters the growth rate were ranged from 0.144±0.22

g to a maximum of 0.24 ±0.38 g day-1. The FCR values ranged from a minimum of

7.3±032 to of 16.1±0.36 and SGR values ranged from a minimum of 0.054±0.001% to

0.091±0.003% of body weight. The FCE values estimated for all the above feeds showing

better FCE of 13.5±0.21 recorded for mussel meal feed formulated with hippa meal as a

source of attractant ie, diet no.3 and remaining diets fed lobsters FCE values were

recorded as 8±0.7, 11.7±0.47, 6.1± 0.35,12.2 ± 0.84, 9.9 ± 0.47 %. Total feed

consumption was recorded as a minimum of 0.006% of body weight per day to a

maximum of 0.017% by individual lobsters.

3.3.9. Biochemical composition

The data on changes in biochemical composition such as protein,

carbohydrate , lipid including moisture content of tested tissues (muscle, gill and

hepatopancreas) of P.homarus fed on experimental diets before and after the

experiments are given in Tables 3.18 to3. 21.

3.3.10. Moisture

In P.homarus (control) at the commencement of experiments the moisture

content in muscle, gills and hepatopancreas was; 80.62 ± 0.21 , 92.52 ±0.86 and 79.52

± 2.48 respectively. In the experimental lobsters the moisture content in all the tested

tissues were considerably decreased over the period. The decrease in variation of

moisture content in muscle tissues of P.homarus were ranged from a minimum of

0.4% to 4.71% and in hepatopancreas and gill tissues the ranges were from 0.6 % to

5.4% and from 4 to 7.6 % respectively (Table 3.18).

The trend observed for the variation in biochemical constituents such as protein,

carbohydrate and lipid contents in the tested muscle, hepatopancreas and gill tissues of

experimental lobsters showed an increase over control values at the termination of the

experiments.For example, the protein content in muscle, hepatopancreas and gill tissues

of P.homarus before the commencement of experiment (control) was 50.64 ±0.84 %,

35.02 ±0.84% and 18.83 ±0.69% dry weight respectively. But the muscle protein content

of experimental lobsters fed with diets 1 to 6 were recorded higher values of 52.41±

0.24%, 52.33±0.83 %, 53.82 ±1.42%, 52.03±0.06%, 50.82±0.19% and 50.63±0.84 % dry

weight in order. Similarly, higher protein content were recorded in the hepatopancreas of

P.homarus fed with experimental diets 1 to 6 were 40±0.38%, 40±0.63%, 42.44±0.86%

38.54±0.94%, 37.64±0.15%, 36.23±0.24%. In the gill tissue also, higher protein content

of 20±0.68%, 20.22±0.44%, 20.44±0.84%, 19.62±0.32%, 19.22±0.31% and

19.04±0.06% were recorded against initial values (Table 3.19).

In general an increase in protein content in all the tested tissues of P.homarus fed

on experimental diets was observed. The increase in protein content ranged from 0.01%

to 3.18% in muscle tissue, 1.21% to 7.42% in hepatopancreas and from 0.21% to 1.61%

in gill tissues.

The initial carbohydrate content in muscle, hepatopancreas and gills of

P.homarus was 7±0.68%, 11.71±0.82 % and 4.03 ±0.82 % dry weight . The carbohydrate

content of the muscle tissue, hepatopancreas and gills tissues was increased from 2.21%

to 5.33%, 0.29% to 2.95% and 0.81 % to 2.83% respectively than control values (Tables

20).

The lipid content in muscle, hepatopancreas and gills of P.homarus (control)

before the commencement of experiments was 8.54±0.86 %, 18.83±0.27% and

4.83±0.62% percentage dry weight respectively. The increase of lipid content in

hepatopancreas was much more obvious than observed in muscle and gill tissues. In the

lobsters fed with diets 1 to 6, the lipid content was ranged from a minimum of 8.63

±0.14% to a maximum of 10.42±0.81 % dry weight in muscle tissues, 4.63 ±1.41 % to a

maximum of 6.03 ± 0.24 % dry weight in gill tissues and 19±0.86 % to a maximum of

22.54 ±0.57 % dry weight in hepatopancreas tissues in order (Table 21).

In general ,the overall increase in lipid content recorded from these selected

tissues showed 0.09% to a maximum of 1.88 % by muscle tissues, a higher percentage of

increase from a 0.17% to a 3.71% in hepatopancreas where as in gill tissues, the lipid

content showed a decrease of 0.2 % and 0.02% recorded from P.homarus fed diet

numbers 5 and 4 respectively and carbohydrate contents in remaining lobsters recorded

increase over control values.

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3.4. DISCUSSION

In feed formulation, water stability of pellets is very important. The stability

mainly depends upon the binders used in the feed. If the pellet is hard, it is difficult for

the lobsters to consume. If it is soft, the pellets would disintegrate faster leading to feed

wastage there by lead to water quality deterioration.

In the present study, when the concentration of tapioca powder increased, it

adversely affected the binding property of the feed pellets . For example , leaching rate

of feed containing tapioca flour added @ 3% were significantly higher than those of the

remaining feeds in which tapioca flour was either not included or added @ 1%. Similar

results were also reported by Ahamad Ali (1988), indicating the binding capacity of

tapioca flour at 2% level was comparable to agar agar, polyvinyl alcohol and sodium

alginate . He further observed that pure water stable starch when added in shrimp feed

even at 40 % was not effective as binder when added as a single source because loss in

weight of pellets was high . Goswamy and Goswamy (1979) also reported similar

observations when starch added as binder in prawn feeds. Ahamad Ali (2005), reported

superior binding properties of maida flour, wheat flour and tapioca flours including

efficiency of wheat flour and guar gum as binders in shrimp feed. However in the

present study, wheat flour and guar gum incorporated feeds were not accepted by the

lobsters. According to Kaladharan, 1998, sea weed meal as a binder in fish and prawn

feed increase physicochemical standards of the feed thereby maintain water quality of

the rearing system . Addition of agar in feeds also helps to retain physical integrity of

the feed particles because of their gelling properties agar is used in food technology. In

the present study , sea weed and agar agar were also added in all the experimental

feeds because of their excellent gelling qualities.

In this experimental studies , out of the 35 test diets analysed , only seven diets

registered palatability with stability. The percentage stability of all the test diets reduced

progressively in accordance to increasing time period and all feeds at all hours of

observation was also showed significant difference when subjected to one way ANOVA.

Stability of the pellets incorporated with different combinations of binders at various

inclusion levels showed binders added in three combinations at 7 % level of inclusion

resulted in the highest stability at a maximum sea water exposure period of eight hours.

Among the diets incorporated with two combinations of binders, the highest stability

was resulted for binders added in combination of agar agar 3% and stick on 1 %

contributing 73.68±3.74 % stability . Binders added in combinations of sodium alginate

(3%), agar agar (3%) and stick on (1%) was also recorded the highest stability

contributing 85.55±5.94 % for 8 hours followed by agar agar (3%), sodium alginate

(1%) and stick on (1%) contributing 84.33 ± 3.74 % stability up to 8 hours of sea water

exposure. The diets added with agar agar @1%, sodium alginate @ 3% exhibited the

lowest stability of 55.80±2.21 % at eight hours of water immersion among the palatable

diets. The diet added with agar at a higher inclusion level of 4 %, with 1 % gelatin, the

feed was rejected by the lobsters on the account of non palatability of gelatin. Though

the combination of binders such as agar agar 3%, stick on 1% and wheat powder 3%

exerted very good stability of 82.21±6.74 %, the feed was traced as unpalatable for

lobsters which is also attributed to the inclusion of wheat powder . Similarly

combination of binders such as agar agar 5%, gelatin 2.5%, stick on 1%,wheat powder 1

%,and guar gum 2.5 when added , even though resulted reasonably good stability for

the pellets contributing 80.65 ±4.46 % , lobsters were found not attracted to the feed

thus indicating unsuitability of gelatin, wheat powder and guar gum for the feed

formulation for P. homarus. The present study clearly showed factors such as quantity

and combinations of the binders, sea water exposure time of the pellets and ingredient

composition of the diet determines the stability and palatability of the diets.

It was also reported that feeds having rough and water soluble ingredients

disintegrates faster (Meyers and Zen-Eldin, 1972). Ahamed Ali,(1998) and Mayers et al.,

(1972 a, b) discussed about the range of substances available as binders. Ahamad Ali,

(1982, 1988, 2010) verified the use of tapioca and other binders viz, agar agar, gelatin,

poly vinyl alcohol, guar gum, polymethylocarbamide, wheat gluten and sodium alginate.

Lamek et al., 2011 reported rapid deterioration of pellet stability at fourth hour onwards

even at a higher inclusion level of binders as much as 33.93 % in the feed. In the present

study, the stability of the pellets gradually decreased at the advancement of time.

According to Anthony Tolomei et al.(2003), attractability of non-soaked diet was greater

than soaked diet and lobsters are capable of detecting the diet regardless of prolonged

exposure to water; however they will selectively move towards a “fresh” rather than a

“stale ’’ diet. Ryther et al.(1988) and Kanazawa,(1994) reported on the physical

requirement of pellet feeds for lobsters. Kanazawa also specified that feed pellets should

be hard and also should be remain stable in water for several hours till consumed,

however the ideal stability requirement of the pellets were not mentioned. Conklin et al.

(1977) developed artificial feed for lobsters and the growth achieved was compared with

lobsters fed brine shrimp. A’abramo et al.(1981) modified the same artificial diet by

incorporating shrimp meal as a major component and achieved growth rate equivalent to

85 % of the brine shrimp standard. D’ Abramo and Sheen,(1994) reported that though

natural food continue to supply micro nutrients, macronutrients become limiting and must

be supplied through prepared feeds.

Ahamad Ali et al.(2010) reported guar gum as a binder in feed formulation of

shrimps. However, in the present study, inclusion of guar gum in the pelleted feed was

found un palatable for P. homarus .The present study recommends, binder combination

at an inclusion level of 3% sodium alginate, 3% agar agar and 1% stick on for the

formulation of artificial diets for sub adults of Panulirus homarus.

According to Aksnes et al. (1997), Opstvedt et al. (2000), Shakila et al.(2003),

inappropriate storage or poor handling of the feed ingredients diminish the quality of the

feed due to bacterial contamination or oxidative decomposition during or after feed

production because they destroys critical nutrients or produce toxins such as biogenic

amines. In the present study, considerable care was taken to preserve the quality of the

ingredients and feed was prepared hygienically during different processing stages . ie

during preparation, handling , drying and storage.

It is well established that the nutrient content of the feed influence growth,

survival and the amount of metabolic and excreted waste products . Processing also plays

critical role as it influences stability of the feed thereby availability of the feed in sea

water over time and all these factors have substantial effect on the amount of waste

produced through pellet fragmentation, leaching loss, residual feed and undigested

material. Feeding strategies have also been found to influence water quality and shrimp

health (Jory,1998; Burford et al.,2001; Cuzon et al.,1982 ). Reports on Soya bean meal

has been used as a suitable substitute for fish meal in diets are available for several

crustaceans such as fresh water shrimp Macrobrachium rosenberghii, Dund Ni, 2003,

and for American lobster Homarus americanus (Floreto et al.,2000), for Australian red

claw cray fish , Cherax quadricarinatu (Gracia et al., 2003). In the present study all the

feeds prepared using soya meal as one of the major ingredients were found accepted by

P.homarus. Since lobsters are bottom dwelling animals, a high density feed is required

for their immediate availability in intact form when introduced in the culture system. In

the present study, all the feeds were of high density and were therefore found as

immediately available to lobsters when introduced in the culture system.

Molting is one of the most important physiological process for crustacean

growth since increase in body size occurs in a series of steps associated with the castings

of the old exoskeleton (Passano,1960). Moulting in crustacean refers to the periodic

shedding of the old confining exoskeleton and the subsequent enlargement of the newly

disclosed integument. Detailed description of moulting process observed for P. homarus

under captivity was given (Radhakrishnan et al., 1998) . In the present study , an uniform

molting rate of two times was observed for all the lobsters fed with diet no 1,2,4,5 and

6; while P. homarus fed with diet no.3 was moulted 3 times. The moult cycle in days

was ranged between 30.30 to a maximum of 45.45 days.

The experimental feeds was also evaluated for their nutritional qualities based on

growth performance of P. homarus determined by body weight gain, survival rate, size

increase, SGR, FCR , FCE etc . The present study showed that out of the 6 experimental

feeds tested P. homarus grown on mussel meal dominated feed performed better than

the remaining feeds added with hippa meal and fish meal in higher concentrations.

Food conversion ratio also showed similar trend. Reports on growth of Homarus

americanus grown over a period of 30 months on dry formulated diets showed a

maximum growth rate of 0.555g day-1 achieved on 53% protein diet (Abramo et

al.,1981).Lucien Brun et al. (1985) reported when juvenile lobsters of H. gammarus fed

with 6 pelletized diets for varying proportions of proteins ranging from 21.8 to 45.8%

resulted linear growth in lobsters fed with feed containing above 35%. Lamek et

al.(2004) reported a growth rate of 0.470g per day for lobsters fed with diets containing

protein levels of 54.9% indicating high level dietary requirement of protein in P.homarus.

The maximum growth of 0.360 g day-1obtained in the present study is slightly lower

than the growth rate reported for P. homarus by Lamek et al.(2004) grown under

experimental culture feeding dry compounded pellet feeds. This study also demonstrated

that mussel meal added feed have greater influence on the growth rate of P.homarus sub-

adults . Radfoerd et al.(2005) reported feeding once in the morning versus once at night

resulted significantly greater growth for J. edwardsii , resulting improved growth rate.

Smith.(2002) reported frequency of feeding did not change the growth rate or survival in

Penaeus monodon. Tolomei et al.(2003) concluded J. edwardsii juveniles need to be fed

once daily. In the present study, the feed was provided as a single dose at dusk daily and

was found consumed by the lobsters soon after their introduction in the culture tanks.

The experimental studies conducted on juveniles of P. ornatus , P. cygnus and

J. edwardsii by feeding pelleted feeds revealed that growth and survival was much

poorer than lobsters fed with mussels (Crearet al., 2000 and 2002; Glencross et al., 2001;

Smith et al., 2003 b) and accordingly concluded that the pellets were either nutritionally

inadequate or not sufficiently attractive to the lobsters. Their studies also revealed that

lobsters cease feeding pellets after one or two hours of introduction in culture tanks;

whereas lobsters consumed mussel flesh even after more than ten hours of sea water

exposure indicating lobsters attractability to live feeds ( Glencross et al.,2001; Tolomei et

al., 2003; Williams et al., 2005b).

An inferior growth of Jasus lalandii juveniles when fed exclusively on pellet

feed was reported by Dubber et al.(2004) than feeding either blue mussel or diet altered

between blue mussels and pellet feeds .The plausible reason quoted for the poor

productivity of Jasus lalandii was low stability of the feed resulting unspecified

nutritional inadequacy. According to Tolomi et al.(2003), superior growth performance

attained by Jasus edwardsii is due to the better nutritional value and digestibility of

mussels and it was not due to the attractiveness of the mussel as evident from the strong

feeding response for formulated feeds exhibited by Jasus edwardsii.

The slow growth of P. homarus on formulated pelleted diets in this study when

compared to higher growth over fresh brown mussel as reported by Lamek et al.(2004)

as well as studies conducted by NIOT through lobster fattening attaining a SGR of

0.407 % body weight per day feeding green mussel Perna viridis indicates formulated

diets used in the present study are only marginally inferior to live mussel.

According to Sheppard et al.(2002) providing a hard pelleted feed should not

be a deterrent to feeding, provided that the feed is perceived by the lobster to be

palatable. According to Smith et al.(2008) the size of the pellet in relation to the size of

the lobster is important to maximize feed consumption and also to minimize feed

wastage. The physical form and size of feed pellets provided to any species of crustacean

have a significant effect on the proportion of the feed item that is ingested by the animal

and accordingly Sheppard et al. (2002) evaluated the efficiency of ingestion of different

feeds such as soft, semi-moist pellets of approximately 1 mm,3 mm and 9 mm in

diameter and also on hard dry pellets for different size groups of Panulirus ornatus .

Based on the studies they concluded that 2 g lobsters feed most efficiently on 1 mm

diameter pellets and larger lobsters consumed 3 mm diameter pellets with significantly

less wastage than on 9 mm pellets. Sheppard et al.(2002) was one who carried out the

first reported systematic study on the optimal feed pellet size for spiny lobster Jasus

edwardsii belonging to three size groups having body weight;14g, 38 g; and 135 g with

three sizes of pelleted feed. They concluded that 38 g lobsters consumed 5x5 mm pellets

and 135 g sized lobsters consumed 7x7 mm pellets most efficiently. In the present study,

the size of 4 mm pellets were found optimum for sub adults of P. homarus because of

their suitability to hold and consume as observed throughout the experimental period.

Tolomei et al.(2003) opined lobsters attraction to shrimp pellets and mussel flesh

declined with increasing immersion time. However, in contrary they further remarked

that feeding shrimp pellets soaked for different time periods ranging from 0.5 to 8 hours

did not negatively affect the growth, feed conversion or survival of the lobsters. In the

experimental growth studies conducted for spiny lobsters feeding diet solely of mussels

showed equivocal results. A number of studies have shown mussel as an excellent source

of food for temperate species such as J. edwardsii, Jasus lalandii and P. cygnus (Crear et

al., 2000 and 2002; Glencross et al., 2001;Ward et al., 2003b; Dubber et al.,2004; and

Srikrishnadhas,1993; Kaleemur,1997 ; Radhakrishnan and Vijayakumaran,1982) for

tropical species. In contrary, Menez et al.(1996) reported thawed green mussel, Perna

viridis was inadequate for the tropical P.ornatus with only 6 % survival by the lobsters

in four months experiments. Similar results were recorded in juveniles of P.ornatus

fed with thawed green lipped and blue mussels i.e. Perna canaliculatus and Mytilus

edulis . According to Smith et al.(2005) and Barclay et al. (2006), though the lobsters

grew well initially , growth and survival rates declined afterwards with mortality

occurring at moulting. They also reported that lobsters gradually became pale in color

finally turning pink tinged exoskeleton when died. P homarus fed with formulated

pelletted feeds in the present study exhibited satisfactory growth with moderately good

FCR and the organoleptic qualities of P homarus was also excellent with their

characteristic pigmentation/ color as observed in natural condition.

The protein requirement for crustaceans have been studied extensively in penaeid

shrimps (Cuzon and Guillamae,1997) and in homarid species (Capuzzo and

Lancaster,1979). However, studies has been focused only on few species of spiny

lobsters such as Panulirus Cygnus (Glencross et al.,2001; P.homarus ( Lamek, 2004 and

Ayyappan, (2002). In the present study , better growth rate was observed in P. homarus

fed with feed no.3 which have higher concentration of protein among other diets. This

can be attributed to the influence of high concentration of mussel meal in the diet leading

to better feed utilization/ conversion and associated growth.

Lamek et al.,(2004), reported P. homarus fed with fresh and live clam attained a

specific growth rate of 0.463 g with a FCR of 8.65 and when fed with crabs, they

attained a specific growth rate of 0.390 g with a FCR of 10.375. According to Simon et

al.,2008, when formulated dry compounded diets containing fresh P. canaliculatus

mussel added in higher increments, showed enhanced protein at 6 % and 10 % of lipid. A

clear response to the protein increments with a maximum growth of 47 % and 53 % was

achieved in juveniles and sub adults of P.ornatus. He also reported about poor growth of

lobsters attained on dry feed which is attributed to the decline in the attractiveness of the

pellets during water immersion. Smith et al. (2005) reported a clear dose response for

increasing dietary protein content and when lobsters feeding highest protein diets

achieved best growth, with a daily growth coefficient of 1.38 % /day.They further

inferred that lobsters require a high protein diet of at least 60 % crude protein. As high

survival and good growth rate within a shortest life span is essential for economic

viability for lobster culture, present study recommends further modification of diet 3

by increasing nutrient contents for the development of an exclusive high quality

formulated diet incorporating higher concentration of mussel meal for spiny lobster

Panulirus homarus .

Documents

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