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
Enhancement of Immune response
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.
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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.
) Pers. mixed diet 43
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 44
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 45
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 46
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 47
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.)
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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.
) Pers. mixed diet 48
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
Enhancement of Immune response
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.
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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.
) Pers. mixed diet 49
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
Enhancement of Immune response
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,
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 50
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 51
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 52
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 53
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
Enhancement of Immune response
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.
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 54
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 55
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
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 56
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,
Enhancement of Immune response
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
nse in Catla catla (Ham.) by Ethanolic extract of Cynodon dactylon (L.) P
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
) Pers. mixed diet 57
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.