CHAPTER ER – 3 - Shodhganga : a reservoir of Indian...

CHAPTER

ER – 3

Enhancement of Immune response

FORMULATI

SUPPLEMENTA

CON

3.1. INTRODUCTION

India is a biodivers

most of which have been

fast developing industry in

contributing fish protein to

source of protein and al

magnesium, sodium, etc.,

and adverse environmenta

losses. Thus, fish farmers h

Prevention of disease is mu

disease process once it be

growing interest in underst

and have at least partial suc

is the important obstacle t

chemotherapeutants and an

for their negative impact l

drug resistance and immun

Development of A

compatible and suitable d

(FCR) and Specific Growt

and suitability of artificia

deoiled cakes and rice bra

feed ingredients, the main

supply and improves the d

normal feed ingredients.

nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P

CHAPTER – 3

LATION OF C. dactylon EXTRACT MIXE

ENTARY DIET AND ESTIMATION OF FO

CONVERSION RATIO IN C. catla

ION

diversity nation and it has a rich background in med

been used to treat human and animal diseases. Aqua

stry in India. Fish farming and aquaculture industries

tein to large Asian population (Ravenhalt, 1982). Fis

and also has essential amino acids with minerals

, etc., (Barlas, 1986). However, unmanaged fish cultu

mental conditions affect the fish health leading to

mers have to carry out careful husbandry practices (S

e is much more desirable than intervention to stop and

e it begins. The recent expansions of aquaculture h

nderstanding fish disease, so that they can be treated o

tial success.The emergence of antibiotic-resistant mic

tacle to their extensive use (Anderson, 1992). Use o

and antibiotics for controlling disease has been wide

pact like residual accumulation in the tissue, develop

mmunosuppression (Anderson, 1992).

t of Aquaculture is mainly depended on the ava

able diet. For the formation of fish diet, Feed Conve

Growth Rate (SGR) are good tools to compute the a

rtificial diet. Normally, balanced fish feeds contai

ice bran. Our research mainly focuses on alternative

main reason is to escalate the cost and uncertainty

the disease resistant capability through prophylactic

) Pers. mixed diet 42

Ch

ap

ter

– 3

IXED

OF FOOD

n medicinal herbs,

. Aquaculture is a

ustries take part in

2). Fish is a good

inerals like zinc,

h culture practices

ing to production

ices (Sakai, 1999).

op and reverse the

lture has led to a

eated or prevented

nt microorganisms

Use of expensive

widely criticized

evelopment of the

e availability of

Conversion Ratio

e the acceptability

contain fishmeal,

rnative sources of

tainty of constant

lactic treatment of


Several ayurvedi

immunomodulators. Many

pathogens and activate the

Karatas et al., 2003). Inste

attention is being paid to th

aquaculture. It may be ach

their chemical component

artificial diet preparation

plant materials have bee

Nandesha, 1990; Ray and

2001). Nowadays, supplem

in aquatic animals. Moreov

study was conducted to eva

growth, survival, body c

activity of experimental fis

3.2. MATERIALS AND

3.2.1. Preparation of Cyn

Previously describe

3.2.2. Experimental diet

Artificial balanced

using fish meal, fish grow

liver oil (Universal medi

vitamin premix (Vetsfar

ingredients (Fig. 3.1, Tab

were designed to prov

concentrations of 0.05%

ethanolic extract of C. dac

normal diet before pellet

ingredients were mixed tho

cold-pelleted with a pelleti

°C. The dried pellets were

containers.


urvedic medicinal plants are acting as a

Many of the herbal plants have the ability to inhibit th

ate the immunity (Immanuel et al., 2004; Chansue

. Instead of vaccination and chemotherapeutic agents

id to the use of immunostimulants for disease control

be achieved only through feed supplement. Medicina

ponents are used as an Immunostimulants, which

ation for aquaculture research and practices. Alrea

e been studied widely for their nutritive values

y and Das, 1992; Harish and Gajaria, 1995 and Nand

pplemental feed treatment is popular for preventing

oreover, they are cheaper, safer and biocompatible.

to evaluate the effects of C. dactylon (L.) mixed artif

ody composition, digestive enzyme activity and

ntal fish C. catla.

AND METHODS

Cynodon dactylon (L.) ethanolic extract

scribed in chapter 2.

l diet preparation

lanced diet was prepared

grower, wheat flour, cod

medicare Pvt. Ltd.) and

etsfarma Ltd.) as feed

, Table 3.1). Diet groups

provide with different

0.05%, 0.5% and 5 %

dactylon mixed with the

pelletization. All dietary

ed thoroughly, moistened,

pelletizer and dried at 40 °C for 24 hrs. Diets were s

were hand crumbled into small pieces and stored in a

Fig. 3.1: Formulated an

feed for exper

C. catla.


Ch

ap

ter

– 3

as a powerful

hibit the microbial

nsue et al., 2000;

agents, increasing

ontrol measures in

edicinal herbs and

hich are used in

Already different

lues (Shetty and

d Nandesha et al.,

nting the diseases

tible. The present

d artificial diet on

and antiprotease

were stored at -20

ed in airtight PVP

ated and pelletized

experimental fish


Table 3.1: Composition of

Ingredients/100 gm

Wheat flour

Dry fish meal

Fish grower

Cod liver oil

Vitamin and mineral pr

Cynodon dactylon extra

(0.05%, 0.5%, 5% level

3.2.3. Proximate compos

Proximate compos

AOAC (2003) is as follows

3.2.3.1. Determina

Moisture was dete

sample was accurately we

allowed in an oven at 10

obtained. Then the crucibl

cooling, it was weighed a

using the formula:

Percentage of m

Wh

Note

3.2.3.2. Determina

For the determenat

furnace at 600 °C for an

crucible was noted (W1). O

sample was ignited over a

the crucible was placed in

gray white ash indicate co


tion of diet Ingredients used for experiments

Experimental diet

(gm)

Contro

(gm

45 45

32 32

11 11

10 10

eral premix 2

extract

level)

Present Abse

mposition analysis

mposition of feed ingredients were analysed by the

ollows.

ination of moisture

s determined by oven drying method. 1.5 gm of

ly weighed in a clean and dried crucible (W1). The c

at 100 – 105 °C for 6 – 12 hrs until a constant

crucible was placed in the desiccators for 30 min to

d again (W2). The percentage of moisture was ca

e of moisture = (W1 – W2 × 100) / (Weight of sample

Where,

W1 = Initial weight of crucible + Sample

W2 = Final weight of crucible + Sample

Note: Moisture free samples were used for further a

ination of ash

rmenation of ash, clean empty crucible was placed

for an hour , cooled in desiccator and then the weig

). One gram of each of sample was taken in crucibl

over a burner with the help of blowpipe, until it is ch

ced in muffle furnance at 550 °C for 2 - 4 hrs. The a

ate complete oxidation of all organic matter in the sa


Ch

ap

ter

– 3

ontrol diet

(gm)

45

32

11

10

2

Absent

by the method of

m of well mixed

The crucible was

stant weight was

in to cool. After

was calculated by

ample)

rther analysis

laced in a muffle

weight of empty

crucible (W2). The

it is charred. Then

The apperance of

the sample. After


ashing, the furnace was s

Percentage of ash was calc

Percentage of ash =

Where,

Diff

3.2.3.3. Determina

Principle

Prepared fish food

were digested by heating w

digestion mixture. The m

formed, released ammonia

against standard HCL. To

nitrogen with appropriate f

Reagents

• 0.1N HCL (stan

• Sodium hydrox

• Digestion mixt

(CuSO4)

• Boric acid: Dis

made the volum

• Indicator: Meth

Procedure

Protein in the sam

dried samples was taken

and 8 gm of digestion m

order to mix the contents

mixture becomes clear (b

was cooled and transferre

mark by the addition of d

Markam Still Distillation


was switched off. The crucible was cooled and we

s calculated using the formula:

sh = (Difference in weight of Ash × 100) / Weight o

Difference in weight of Ash = W3 – W1

ination of protein

food protein was determined by Kjeldahl method. T

ating with concentrated sulphuric acid (H2SO4) in the

he mixture was then made alkaline. Ammonium su

monia which was collected in 2% boric acid solution

L. Total protein was calculated by multiplying the

riate factor (6.25) and the amount of protein was calcu

L (standard), Concentrated sulphuric acid

ydroxide solution 40% w/w

mixture: Potassium sulphate (K2SO4) and copp

d: Dissolved 40 gm of boric acid in sufficient distille

volume up to 100 ml.

: Methyl red

e sample was determined by Kjeldahl method. 0.5

taken in digestion flask. Add 10 – 15 ml of concentr

tion mixture i.e. K2SO4:CuSO4 (8:1). The flask wa

ntents thoroughly then placed on a heater to start dige

lear (blue green colour) and it took 2 hrs to complete

nsferred to 100 ml volumetric flask and volume was

n of distilled water. Distillation of the digest was p

llation Apparatus (Khalil and Manan, 1990). Ten m


Ch

ap

ter

– 3

nd weighed (W3).

eight of sample

thod. The samples

in the presence of

ium sulphate thus

lution and titrated

ng the amount of

s calculated.

copper sulphate

distilled water and

. 0.5 – 1.0 gm of

ncentrated H2SO4

sk was swirled in

rt digestion till the

plete. The digest

e was made up to

was performed in

Ten milliliters of


digest was introduced in

gradually added through t

NH3 produced was collec

boric acid solution with

distillation, yellowish col

against standard 0.1N HC

also run through in all abo

was calculated by using th

Percentage o

%N = [(S - B) ×

Whe

3.2.3.4. Determina

Dry extraction met

extracting dry sample with

phospholipids, sterols, fa

extracted together. Therefo

was determined by inte

determined by ether extrac

moisture free sample was

introduced in the extractio

was filled with petroleum

start the extraction. After 4

the beaker before last siph

ether washing and evapora


ced in the distillation tube then 10 ml of 0.5 N

ough the same way. Distillation continued for at least

collected as NH4OH in a conical flask containing 2

n with few drops of modified methyl red indica

sh colour appears due to NH4OH. The distillate was

N HCl solution till the appearance of pink colour.

all above steps. Percentage of crude protein content o

sing the formula:

tage of protein = 6.25* × %N (* Correction factor)

B) × N × 0.014 × D × 100] / Weight of the sample ×

Where,

S = Sample titration reading

B = Blank titration reading

N = Normality of HCl

D = Dilution of sample after diges

V = Volume taken for distillation

0.014 = Milli equivalent weight of Ni

ination of Crude fat

n method for the determination of fat was implied.

le with some organic solvent, since all the fat materi

ls, fatty acids, carotenoids, pigments, chlorophyll,

herefore, the results were frequently referred to as cr

intermittent Soxhlet extraction apparatus. Crud

extract method using Soxhlet apparatus. Approximate

was wrapped in filter paper, placed in fat free thimb

traction tube. Weighed, cleaned and dried the recei

leum ether and fitted in to apparatus. Turned on wa

After 4 - 6 siphoning allow the ether to evaporate and

st siphoning. The extract is transferred into clean gla

aporated ether on water bath. Then, the dish is placed


Ch

ap

ter

– 3

.5 N NaOH was

t least 10 min and

ning 20 ml of 4%

indicator. During

e was then titrated

lour. A blank was

tent of the sample

ple × V

r digestion

lation

of Nitrogen

plied. It contained

materials e.g. fats,

phyll, etc., were

o as crude fat. Fat

Crude fat was

ximately, 1 gm of

thimble and then

receiving beaker

on water heater to

ate and disconnect

an glass dish with

placed in an oven


at 105 °C for 2 hrs and c

determined by using the fo

Percentage of crude

3.2.4. Experimental anim

Similar age groups

carp, Catla catla was obtai

fish farm (Fig. 3.2), Karan

Tamil Nadu, India. Averag

(Fig. 3.3d) used for the

88.05 ± 4.75 gm. All fish

in fiber reinforced plastic (

3.3a, b and c). The water w

in two days to maintain t

Borewell water was used

with 2 hrs aeration daily. D

dissolved carbon dioxide

alkalinity of water wer

weekly intervals (APHA,

kept at the ambient, unc

photoperiod. Fish were acc

diet prepared in the laborat

a

c


Fig. 3.2: Panoramic view o

Golden fish farm

and cooled it in a desiccator. The percentage of cr

the following formula:

rude fat = Weight of ether extract × 100 / weight of s

l animal and their maintenance

groups of Indian major

s obtained from Golden

Karandhai, Thanjavur,

Average weight of fish

r the experiment was

ll fish were maintained

lastic (FRP) tanks (Fig.

ater was replaced once

tain the water quality.

used to rear the fish

aily. Dissolved oxygen,

ioxide, pH and total

r were monitored at

PHA, 1985). Fish were

t, uncontrolled temperature of 28 ± 2 °C under

ere acclimated for 15 days and fed ad libitum with b

aboratory.

Fig. 3.3: Experimental aquarium in th

(a) Stock c

(b) Experi

(c) Tank w

(d) Catla c

b

d


Ch

ap

ter

– 3

view of Karandhai

farm

of crude fat was

ht of sample

under the natural

with balanced fish

in the laboratory

tock culture tanks

xperimental tanks

ank with fish

tla catla (Hams.)


3.2.5. Feeding trial

The feeding trial w

fed with apparent sanitatio

experimental period. Exper

2 % of their body weigh

namely, diet group I (DG1

IV (DG4) and they were f

respectively.

The initial body we

weighed randomly at 10 d

to determine average read

The morphologica

1. Specific growth rate (

where,

Log

Log

2. Feed conversion ratio (F

3. Average daily growth (A

On the termination

weighed individually and a

analysis of protein, carboh

the methods of AOAC (200

3.2.6. Digestive enzyme a

Hepatopancreas of

for the measure of dig

9 AM to 11 AM for the rem

kept in frozen condition at


trial was conducted over a period of 45 days. The tes

nitation twice a day at 9 AM and 6 PM during the

Experimental fish were fed with supplementary diet a

weight per day. Fish were randomly divided into f

(DG1), diet group II (DG2), diet group III (DG3) an

were fed with 0, 0.05%, 0.5% and 5 % plant extract

dy weights of each fish were recorded. Fish from eac

t 10 days of interval. The experiment was conducted

readings.

logical growth parameters were calculated as follo

rate (SGR) (% day-1

) = (Log Wt – Log W0 × 10) / Fe

Log Wo: weight of fish on the first day of trial,

Log Wt: weight of fish on the last day of trial.

ratio (FCR) = Dry weight of feed (g) / Live weight gai

wth (ADG) = (Final body weight – Initial body weigh

fee

nation of the experiment, all the surviving fish wer

and a portion of the dermal muscle was dissected fo

carbohydrate, moisture, ash and fat content of the m

C (2003).

yme activity

eas of fish were collected every 10 days of experim

f digestive enzyme activity. Fish were taken

the removal of hepatopancreas. All removed hepatopa

ion at -70 °C until enzyme assays were conducted.


Ch

ap

ter

– 3

he test diets were

ng the 45 days of

diet at the rate of

into four groups,

3) and diet group

extract mixed diet

m each tank were

ucted in triplicate

s follows:

0) / Feeding days

ht gain (g)

weight) / No. of

feeding days

were harvested,

cted for proximate

the muscle as per

perimental period

aken at morning

patopancreas were


Approximately 1.0

Tris–HCl buffer at pH 7.5

10,000 × g for 30 min a

duplicate. Total soluble pr

(total protein and albumin

Pharmaceutical Ltd). Amy

using soluble starch as th

protease activity was as

modification, using casein

activities were measure

UV - visible spectrophotom

3.2.6.1. Amylase a

Amylase activity

mixture containing 0.5 m

prepared in 0.2 M acetate

was incubated at 50 °C. A

adding 1.0 mL of DNS (3,

20 mL of 2 M NaOH, to w

filled with water to 100 m

cooling, 18 ml of water wa

enzyme activity was defin

reducing sugar (glucose) in

3.2.6.2. Protease a

Activity for neutra

some modifications. Hepa

5.0 ml substrate (0.45% ca

incubation, Trichloro ace

This mixture was centrifug

of released amino acids we

presence of the Folin-Cioc

range 0.02 - 0.24 µmol/ml.


ly 1.0 gm of hepatopancreas were homogenized in chi

H 7.5 and enzyme extracts were obtained after centr

min at 4 °C. The supernatant of each sample was

ble protein was measured by commercially available

bumin kit, Qualigens Diagnostics, division of Glaxo

. Amylase activity was assayed by the Bernfeld met

as the substrate and react with 3,5-dinitrosalicylic

as assayed according to Anson (1938) method

casein as the substrate and react with Folin reage

easured as there is a change in absorbance

photometer (UV-2310 Techcomp, China).

se assay

ivity was measured by Bernfeld method (1955).

0.5 ml of 1 % (mass per volume ratio) soluble sta

cetate buffer and 0.5 ml of appropriately diluted enzy

°C. After 10 min of incubation the reaction was te

3, 5-dinitrosalicylic acid) solution (1 gm of DNS

, to which 30 gm of sodium potassium tartarate wer

100 ml). Reaction mixtures were boiled for 15 m

ter was added. Absorbance was measured at 540 nm.

s defined as the amount of enzyme that released is

ose) in 1 min under the assay conditions.

se assay

neutral protease was determined by Anson method

Hepatopancreatic centrifuged solution (0.5 ml) was

5% casein in 50 mM Tris-HCl buffer) at 50 °C for 1

aceticacid (TCA, 110 mM) was added to attenuate t

ntrifuged at 10000 × g for 5 min and this provides a

ids were measured at OD 670 nm, to one µmol of tyr

Ciocalteau reagent by using a tyrosine standard cu

ol/ml.


Ch

ap

ter

– 3

in chilled 10 mM

r centrifugation at

e was assayed in

ailable protein kit

Glaxo Smithkline

ld method (1955),

licylic acid. Total

thod with slight

reagent. Enzyme

rbance using a

955). A reaction

le starch solution

d enzyme solution

as terminated by

DNS dissolved in

te were added and

15 min and after

0 nm. One unit of

is 1 mmol of

ethod (1938) with

) was mixed with

for 10 min after

nuate the reaction.

ides a colouration

of tyrosine, in the

ard curve over the


One unit of prote

1.0 µmol (181 µg) of tyro

was expressed as U/ml/min

3.2.7. Immunization of fi

Another set of expe

the rate of 2 % of the body

of RaRBC (Appendix II.a)

to the fish using 1 ml tuber

3.2.8. Collection of blood

After immunizatio

experimental period. For b

and were bled from comm

24 G needle (Michael et al

200 µl of blood was draw

1 min. The blood was co

temperature. The clot was

by aspiration was stored in

3.2.9. Assay of serum ant

Serum anti-protease

20 µg of trypsin dissolved

100 µl of Tris-HCl was add

the positive contro, no ser

of Tris- HCl and incubated

0.1 mM substrate BAPN

chemicals), was dissolved

added to all tubes and aga

the reaction was stopped by

measured at 410 nm by u

China). The percentage of

Chakrabarti (2004):

Tryp

where,


protease hydrolyzed casein to produce colour eq

f tyrosine per minute at pH 7.5 at 50 °C. ‘The enzy

ml/min’.

n of fish

f experimental diet groups were fed with the supplem

e body weight for 30 days. On 5th

day, 500 µl of 20 %

x II.a) in phosphate buffered saline was injected intra

l tuberculin syringe fitted with 28 G needle.

blood samples

ization, blood was collected at 7th

, 14th

, 21st and

. For bleeding, each fish were individually caught usi

common cardinal vein using 1 ml tuberculin syringe

et al., 1994). In order to sample the blood for serum

s drawn and whole bleeding procedure was compl

as collected in serological tubes and allowed to c

t was then spun down at 400 × g for 10 min. The seru

red in sterile eppendorf tubes at -20 °C for further use

m anti-trypsin activity

rotease activity was performed by incubating 10 µl of

solved in 100 µl of Tris-HCl (50 mM, pH 8.2). In s

as added to 10 µl of serum, instead of trypsin in Tris

no serum was added to trypsin. All tubes were filled

ubated for 1 hr at room temperature. After the incubat

BAPNA (Na-benzoyl-DL-arginine-p-nitroanilide H

solved in Tris-HCl (containing 20 mM calcium ch

nd again incubated for further 15 min. At the end of

ped by adding 500 µl of 30% acetic acid. The optical

by using UV-Visible spectrophotometer (UV-2310

age of trypsin inhibition was calculated as described

Trypsin inhibition (%) = (A1 - A2/A1) ×100


Ch

ap

ter

– 3

our equivalent to

e enzyme activity

pplemental diet at

f 20 % suspension

d intraperitoneally

and 28th

day of

ht using a dip net

yringe fitted with

serum separation,

completed within

d to clot at room

e serum collected

er use.

µl of serum with

). In serum blank,

n Tris-HCl, and in

filled with 200 µl

ncubation, 2 ml of

lide HCl, Sigma

um chloride), and

end of incubation,

ptical density was

2310 Techcomp,

ribed by Rao and


A1

A2

3.2.10. Statistics

Statistical analysis

for all the parameters w

n = 6 fish/group. All treatm

3.3. RESULTS

Preparation of diet

proximate composition of

DG4) is summarised in Ta

almost equal to the con

experimental diets do not

increased marginally. Ash

to the reduction of percen

Various feed conversion p

compared to diet groups,

weight of DG3 and DG4. H

DG1 and DG2. Net and av

groups. Specific growth ra

Even though no significan

groups, noticeable surviva

control group indicated th

them were acceptable. Acc

in Table 3.4 showed no si

were found among the fou

all C. dactylon incorporate

the moisture and ash conte

be significantly (P < 0.05)

Specific enzyme ac

every 10 days of interval in

of amylase and protease


1 = control trypsin activity (without serum)

2 = activity of trypsin remained after addition of se

alysis is to compare the mean differences among each

ters was computed by student t- test at P <

treatments were assayed in triplicate.

f diet for the experiment is given in Table 3.1 and the

on of the feed ingredients used in the trial (DG1, DG

in Table 3.2. The nutritional profile of C. dactylon

e control feed ingredients. Lipid level of the c

o not differ largely, but crude protein content in th

. Ash content in the DG1 diet was lower than DG4, p

percentage of C. dactylon extract mixed in the con

sion parameters were studied and described in Table

oups, a significant difference (P < 0.05) was obser

G4. However, there were no significant difference fo

and average weight gains are higher in DG3 and DG

wth rate linearly increased in the C. dactylon mixed

ificant difference (P < 0.05) was found among the e

urvival rate of fish (SR100%) in all experimental

ted that the culture condition and composition diets

e. According to the results of proximate body compo

no significant differences (P < 0.05) on body moist

he four groups. Increased level of protein and lipid w

porated diet groups than DG1, while slight variations

content. However, crude protein and lipid content w

0.05) different among the experimental groups.

me activities of amylase and protease activity were

rval in C. dactylon mixed diet treated C. catla. Speci

otease activity were significantly (P < 0.05) hi


Ch

ap

ter

– 3

n of serum

g each diet groups

< 0.05 level,

nd the percentage

1, DG2, DG3 and

mixed diet is

the control and

t in the DG4 diet

G4, probably due

e content of diet.

Table 3.3. When

observed in final

ence found among

d DG4 than other

ixed diet groups.

the experimental

ental groups and

diets provided to

composition given

moisture and ash

ipid were found in

ations observed in

tent were found to

were recorded at

Specific activities

5) higher in all


experimental diet group th

DG4, both protease (Fig.

(P < 0.05) higher at 30 day

of 40 days of experimenta

though, experimental grou

The trend of dominating s

C. dactylon mixed feed wa

Fig. 3.4: Specific activit

gland of C. catl

The values represe

Statistical differenc

significant differenc

DG3 and DG4 are

The antiprotease a

control group throughout

DG2, DG3 and DG4 wer

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

Prote

ase

act

ivit

y (

IU/m

g)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Am

yla

se a

ctiv

ity (

IU/m

g)

a

b


oup than the control diet throughout the experimenta

(Fig. 3.4a) and amylase (Fig. 3.4b) activity were s

30 days of feeding than DG1, DG2 and DG3. Wherea

mental period, all groups of enzyme activity were red

l group of DG2, DG3 and DG4 were slightly higher

ating specific amylase and protease activity in C. ca

ed was continued till the end of 40 days.

activities of enzymes (a. Protease and b. Amylase)

catla fed with different experimental diets.

represented were the mean ± SE of 6 fish/group (serum assaye

differences (P < 0.05) among groups are indicated by differe

difference appears among the groups marked with the same lette

4 are represented diet group I, II, III and IV respectively.

ease activity in serum of test groups exhibited mo

hout the study period (Fig. 3.5). Antiprotease activ

4 were significantly (P < 0.05) higher during all e

a

aa

aab

ab

a

abab

ab

ab

ab

b

bb

b

10 20 30 40Days of feeding

DG1 DG2 DG3 DG4

a

aa

a

a

a

aba

ab

abab

abb

bb

b

10 20 30 40

Days of feeding

DG1 DG2 DG3 DG4


Ch

ap

ter

– 3

imental period. In

were significantly

hereas, at the end

reduced. Even

higher than DG1.

. catla fed with

lase) in digestive

ssayed in triplicate).

different letters. No

e letter. DG1, DG2,

ed more than the

activity of DG1,

all experimental


days. From first and seco

serum antiprotease activi

(21st and 28

th day respectiv

in almost all experimental

C. dactylon plays a vital ro

fish can defend more stron

Fig 3.5: Effect of serum

The values represen

Statistical differenc

significant differenc

DG3 and DG4 are r

a

65

70

75

80

85

90

Per

centa

ge o

f T

ryp

sin

Inhib

itio

n


second collection (7th

and 14th

day respectively) o

activity was slightly higher than third and fourth

spectively). DG3 and DG4 maintained their antiprote

ental days. Present study revealed that the experime

ital role in enhancement of serum antiprotease activit

strongly against invading pathogens.

erum trypsin inhibition after immunization with rabbi

resented were the mean ± SE of 6 fish/group (serum assayed

fferences (P < 0.05) among groups are indicated by differe

fference appears among the groups marked with the same lette

4 are represented diet group I, II, III and IV respectively.

aa

a

ab

abab

ab

b abab bb

b

b

7 14 21 28

Days after Immunization

DG1 DG2 DG3 DG4


Ch

ap

ter

– 3

ely) of blood, the

fourth collection

tiprotease activity

perimental diet of

activity. Thus, the

rabbit RBC.

ssayed in triplicate).

different letters. No

e letter. DG1, DG2,

b


Table 3.2: Prox

Composition

Moisture

Crude protein

Lipid

Ash

The values represented

DG1, DG2, DG3 and D

Table 3.3: Feed conversi

with C. dactyl

Parameters DG

Fish no. 20

IBW 88.05 ± 4

FBW 94 ± 4.69

NWG 5.95 ± 0.0

ADG 0.12 ± 0.0

SGR 0.13 ± 0.0

FCR 0.51 ± 0.0

SR 100

The values represented were t

superscripts are significantly dif

I, II, III and IV respectively. IB

weight gain (g), ADG––average

conversion ratio (g), SR–– survi

Table 3.4: Proximate bod

diets (%)

Composition DG

(%

Moisture 75.44 ±

Crude protein 68.65 ±

Lipid 5.74 ± 0

Ash 13.86 ±

The values represented were t

superscripts are significantly dif

I, II, III and IV respectively.


Proximate composition of the experimental diets (%

DG1

(%)

DG2

(%)

DG3

(%)

DG4

(%)

6.92 7.03 7.06 7.08

41.42 41.86 42.85 44.15

5.99 6.32 6.45 6.48

12.42 12.63 12.84 13.04

sented were the mean ± SE of 6 fish/group (in triplicate).

3 and DG4 are represented as diet group I, II, III and IV respecti

nversion and morphological parameters of C. catla aft

ctylon supplemented feeds for 45 days

DG1 DG2 DG3

20 20 20

05 ± 4.75 88.05 ± 4.75 88.05 ± 4.75 88

± 4.69 93.95 ± 4.67 100.15 ± 2.4 10

5 ± 0.06 5.9 ± 0.11 12.1 ± 0.58 12

2 ± 0.001 a 0.12 ± 0.001

a 0.25 ± 0.04

b 0.2

3 ± 0.02 a 0.13 ± 0.02

a 0.27 ± 0.5

b 0.3

1 ± 0.04a 0.58 ± 0.02

b 0.66 ± 0.07

c 0.6

100 100 10

were the mean ± SE of 6 fish/group (in triplicate). Values

ntly different (P < 0.05). DG1, DG2, DG3 and DG4 are represen

ly. IBW––initial body weight (g), FBW––final body weight (g

verage daily growth (g), SGR–– specific growth rate (% day-

survival ratio (%).

te body composition of C. catla fed with different exp

DG1

(%)

DG2

(%)

DG3

(%)

.44 ± 0.10 75.34 ± 0.12 75.95 ± 0.16 75.9

.65 ± 0.25 a 71.56 ± 0.12

b 72.68 ± 0.18

c 73.4

74 ± 0.12 a 6.32 ± 0.05

b 6.66 ± 0.12

b 7.21

.86 ± 0.12 14.36 ± 0.10 14.05 ± 0.12 14.6

ere the mean ± SE of 6 fish/group (in triplicate). Values

ntly different (P < 0.05). DG1, DG2, DG3 and DG4 are represen


Ch

ap

ter

– 3

ets (%)

DG4

(%)

7.08

44.15

6.48

13.04

spectively.

after feeding

DG4

20

88.05 ± 4.75

102.7 ± 2.51

12.65 ± 0.43

0.29 ± 0.04 b

0.33 ± 0.46 c

0.68 ± 0.07 c

100

alues with different

presented diet group

ight (g), NWG––net -1

), FCR–– feed

ent experimental

DG4

(%)

75.96 ± 0.26

73.44 ± 0.18 d

7.21 ± 0.12 c

14.68 ± 0.10

alues with different

presented diet group


3.4. DISCUSSION

The results of the p

supplementation enhances

levamisole supplementatio

Arul, 2006). On the contra

chitosan at 2%, 5%, and 10

et al., (1987) experimente

chitin, chitosan or cellulos

and yellow tail. In our res

were increased in the C.

Hepher (1978) proved that

conventional diets. Poss

increased significantly (P

diets. Likewise, increase

fingerlings fed with Cald

may be higher due to lysin

FCR in fish fed with DG3

ingredient in C. catla by fi

digestibility (Rubanza et

DG3 and DG4 of exper

experimental diet groups, w

experiment. The significa

experimental groups can b

in C. dactylon (Stewart, 19

body composition, and d

vannamei by treating with

body condition scores an

could also be due to high d

our study, the diet compo

diet groups were relative

groups. In the proximate v

DG3 and DG4 than othe

amount of spirogyra incorp


f the present study clearly show that dietary C. dact

ances the growth of C. catla. Similarly, dietary c

ntation enhances the growth of common carp (Gopala

contrary, depressed growth in tilapia after feeding wit

and 10% level were observed by Shiau and Yu (1999

imented that the feeding of supplemented diet cont

ellulose do not affect the growth of red sea bream, J

our results, net weight, average weight and specific

C. dactylon mixed diet groups. Similarly, San

ed that the better growth of fish fed on algae enriche

Possibly, the presence of herb C. dactylon, FCR

P < 0.05) in C. catla between the control and e

creased FCR value found in Sarathrodon nilot

aldophora glomerata incorporated experimental

o lysine content in the alga (Appler and Jauncey, 1983

h DG3 and DG4 diets could also activate the absorp

by fibre rich herbs. Fibre fraction defines extent and

et al., 2005). SGR and FCR were significantly i

experimental diet groups and attained 100% of

oups, which proved that the composition of diet is suit

nificant increase in SGR, FCR and 100% survival

can be attributed to the presence of higher essential

art, 1973). Significant responses were found in survi

and digestive enzyme activity of white shrimp

with medicinal herbs and Bacillus (Ming-Chao Yu,

es and average daily growth observed in supplem

high dietary protein and energy intake (Rubanza et a

omposition of crude protein and lipid levels in all e

latively there was no significant variations found

ate value of crude protein and lipid composition we

n other diet groups. Likewise, direct relationship b

incorporated diet, and muscle protein and fat content


Ch

ap

ter

– 3

dactylon extract

tary chitosan and

Gopalakannan and

ng with chitin and

(1999) and Kono

t containing 10%

eam, Japanese eel

ecific growth rate

, Sandbrook and

nriched diets than

, FCR value also

and experimental

iloticus tilapia

ental diet, which

, 1983). The high

absorption of diet

nt and rate of feed

antly increased in

% of SR in all

is suitable for this

rvival rate in the

ential amino acids

survival, growth,

rimp Litopenaeus

o Yu, 2009). High

pplemented steers

et al., 2005). In

n all experimental

found in the diet

ion were higher in

ship between the

ontents in C. catla


were demonstrated by H

important source of energy

5% Ulva meal at low a

performance, feed efficie

tilapia (Ergün, 2008). Opt

conversion ratios, nutrient

2007).

C. dactylon mixed d

activity of amylase and

resulted in an increase sp

digestive gland (Ming-Ch

enhancement in growth p

fish. The noticed changes

increased absorption of fee

fish. Medicinal herbs con

digestive processes by en

diminishing digestibility of

Principally, α1 pr

restricting the ability of ba

from pathogenic organis

experimental diet containin

C. catla. C. dactylon play

in all experimental days.

Chakrabarti (2004) that th

mixed diet for 4 weeks en

provide the resistance agai

et al., 1999, plasma bac

considerably increased

Scophthalmus maximus. M

was high in serum by im

aqueous extract of Eclipta

weeks of feeding in Oreoc


by Harish et al., (2004). Dietary lipids in aquafe

energy and essential fatty acids (Sargent et al., 2002).

low and high lipid levels significantly improves

efficiency, nutrient utilization, and body compositi

). Optimum lipid levels results in improved growth

utrient utilization and reduced nitrogen excretion (M

ixed diet treated C. catla exhibited the increased leve

and protease. Administration of Bacillus bacteria

ase specific activity of amylase and protease in t

Chao Yu, 2009). Our results were also foun

wth parameters and specific activities of digestive

anges in digestive enzymes may lead to enhanced di

of feed, which in turn contributed to the progression i

bs contain potent bioactive substances, which ma

by enhancing or impairing enzyme activity and im

ility of nutrients (Lin et al., 2006).

α1 protease inhibitor and α2 macroglobulin play

of bacteria to invade and grow in fish by acting again

rganisms (Ellis, 2001). The present study revea

ntaining C. dactylon has developed the non specific i

play a vital role in enhancement of serum antiprote

days. This is an agreement with the observation

that the feeding of C. catla with Achyranthes asp

eks enhanced the level of serum antiprotease level, w

e against the bacterial pathogens. Experimental resu

a bactericidal activity and total protein concentr

sed by oral administration of oxytetracycline

. Magnadottir et al., (2006) stated that the antiprote

by immunization or infection. Similar result was al

ipta alba, increased the serum antiprotease activity

eochromis mossambicus (Christybapita et al., 2007


Ch

ap

ter

– 3

aquafeeds are an

002). Inclusion of

roves the growth

position of Nile

rowth rates, feed

on (Martins et al.,

d level of specific

cteria to shrimps

e in the shrimps’

o found that the

stive enzymes of

ced digestion and

ssion in growth of

h may influence

and improving or

play a role in

against proteases

reveals that the

ecific immunity in

tiprotease activity

ation of Rao and

aspera (0.5 %)

evel, which might

l result of Tafalla

ncentrations were

ycline to turbot

tiprotease activity

as also found in

ctivity after 2 or 3

, 2007). Similarly,


the antiprotease activity e

taxanthin, a carotenoid f

4 months (Thomson et al

protease inhibitor levels w

fish can defend more stron

The results of the p

weed, can serve as an al

deoiled groundnut cake,

disease management for t

feed for C. catla. Howeve

trials to assess the potentia


vity enhanced in rainbow trout by experimental die

noid from natural source (Carophyll pink) supple

al., 1995). When fish were fed with C. dactylon mi

vels were enhanced in C. catla. Thus, the results revea

strongly against invading pathogens.

f the present study clearly demonstrate that C. dactylo

an alternative replacement to the costly feed ingr

cake, jowar powder and reduce the additional exp

t for the vaccine or immunomodulator/immunostimu

owever, complete investigation is required in long-

tential of C. dactylon and optimum dietary inclusion l


Ch

ap

ter

– 3

tal diet containing

supplemented for

mixed diet, the

s revealed that the

ctylon, a natural

d ingredients like

al expenditure in

ostimulator in the

-term feeding

usion levels.

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