International Journal of Aquaculture, 2013, Vol.3, No.16, 85
-
91
http://ija.sophiapublisher.com
89
probiotics have appeared to improve the digestion of
protein, starch and fat that could be due to higher
level of enzyme activities, which may explain the
better growth and feed utilization. Notwithstanding
all these findings, the relationship between digestive
enzyme activities and feeding habits in fishes is still
not very clear.
The physicochemical parameters have an integral role
in the life of fish and fluctuations in their values
adversely affect the health and growth of fish
(
Soderherg, 1990). Throughout the experimental trial
all the water quality parameters remained within the
acceptable range due to constant flow and exchange of
water in fish rearing tanks.
3
Materials and Methods
3.1
Experimental Fish Species
Locally available fingerlings of
Labeo rohita,
Cirrhinus mrigala, Catla catla
and
Hypophthalmicthis
molitrix
of uniform age
and size were collected from
commercial rearing units and transferred to cemented
circular tanks for acclimatization.
3.2
Experimental Design and Protocol
Fishes were stocked in glass aquaria of size (3×2×2 ft)
with 250 L water holding capacity in each. There were
2
treatments (fish species vs. feed ingredients) with 4
and 3 levels in each respectively. Each treatment level
was replicated. Rice polish served as control. Before
stocking, all the aquaria were well cleaned with tube
well water and then disinfected with 700 ppm KMnO
4
solution. Ten fishes of each species were then
randomly selected from the bulk stock and were
weighed and measured. Aquaria were then filled with
fresh tap water up to 1.5 ft level. After acclimatization
fishes were randomly harvested from the main stock
and transferred to individual aquarium @ 10 fish per
aquaria of each species in the relevant replica assigned
to each treatment. Average weight and length of
Labeo rohita
,
Cirrhinus mrigala
,
Catla catla
and
Hypophthalmicthys molitrix
were 3.12g, 7.5cm; 3.18g,
7.0
cm; 3.98g, 10.0cm and 3.2g, 7.0cm respectively.
All the four species were split into four groups in such
a way that each species receives single ingredient at
the same time. After the completion of experimental
trial fishes were randomly collected from each
treatment, 2 fish from each replicate. Sample of each
species were weighed and measured and then belly
was excised. Gut was removed and wrapped up in
aluminum foil and froze at 1
℃
for safe extraction of
digestive enzymes. Water from each aquarium was
totally drained off and remaining fish stock was totally
harvested, weighed and measured and then released
into ponds.
3.3
Enzyme Extraction and Quantitative Estimation
The stored samples were transported in sample tubes
to Biochemistry laboratory for extraction of digestive
enzymes (amylases, lipases and proteases). The gut of
each fish was collected in micro-centrifuge tube and
homogenized with chilled tris HCl in homogenizer.
Homogenate was centrifuged at 6000×g at 4
℃
for 15
min; supernatant was collected and stored at 0
℃
for
enzyme estimation.
3.3.1
Amylase
Starch was used as the substrate in the determination
of amylase activity (Bernfeld, 1955) where 1ml of
properly diluted gut extract was incubated for 3 min at
37
℃
with 1ml of 1% starch substrate (1 g soluble
starch and 0.0067 M NaCl in 100 ml 0.02 M NaH
2
PO
4
,
pH 6.9). The reaction was stopped by the addition of 2
ml 3.5- dinitrosalicylic acid reagent. The solution was
then heated for 5 minutes in boiling water, cooled and
20
ml distilled water was added. The absorbance at
540
nm was read and a standard curve was plotted
with maltose (0.1 mg/mL~1.0 mg/mL distilled water),
to convert readings into mg of maltose( The specific
activity of amylase is defined as 1 mg of maltose
produced minute
-1
mg
-1
protein at 37
℃
).
The amount
of soluble protein in the gut extracts was determined
by the Lowry method (Lowry et al., 1951) using
bovine serum albumin as a standard protein. 0.1 mL of
gut extract sample was added to 0.1 mL of 2 N NaOH.
The mixture was then hydrolyzed at 100
℃
for 10
minute in boiling water bath, then cooled to room
temperature and 1 ml of freshly mixed complex-forming
reagent was added. After 10 minutes, 0.1 mL of Folin
reagent was added and mixed using a vortex mixer.
The absorbance was then read at 550 nm after 30
minutes.
Spectrophotometer was set at 540 nm wavelength
level for assessment of absorption level of blank
and standard. Samples were taken in cuvett tubes
and turned the blank to 0. Again readings of each
sample were taken on spectrophotometer and