Abstract

A feeding trialwas conducted to determine the effect of exogenous enzyme (amylase)

supplementation on growth, apparent nutrientdigestibility, digestive enzyme activity and body

composition of Labeo rohita for 90 days. Labeo rohita fingerlings were allocated to two

treatments and one control diet along with replica of each. Prior to experiment fingerlingswere

acclimatized at laboratory conditions with control diet having 32% of crude protein. Diet was

given at the rate of 4% of live wet body weight once a day. Physical parameters of water like

temperature, dissolved oxygen and pH were monitored by using YSI pro series multipara meter

professional plus meter. Dissolved oxygen was maintained at 5-7 ppm by using air pumps

through capillary system. Metamorphic attributes of fingerlings like length and weight were

measured after intervals of every 15 days to assess growth rate of Labeo rohita. Fecal material

was collected on daily basis for further digestibility analysis. After the completion of 90 days’

trial, fish body meat was analyzed for different nutrients. Then at the end of trial data on growth,

nutrients apparent digestibility and body composition was further subjected to statistical analysis

for finding out the effect of enzyme Amylase. The results revealed that supplementation of

enzyme amylase play a pivotal role in growth and digestibility ofLabeo rohita. Different level of

enzyme application showed noticeable effect on body composition and enzyme activity of rohu.

Key words:

Labeo rohita, exogenous enzyme, apparent nutrient digestibility, growth, body composition
Chapter 1

Introduction

Aquaculture has risen enormously over the last three decades, with an average annual
growth rate of 6.9%. It is now the fastest-growing sector of the global food system. The lengthy
history of land-based pond culture of fish and shrimp in fresh and brackish water in Asia has
contributed significantly to the region's output dominance in the aquaculture business (Ottinger
et al., 2022).Aquaculture accounts for 46.0 percent of total global fisheries landings and
production (178.5 million tonnes; FAO 2020).Aquaculture centers are experimenting with
different culture technologies and sites in order to meet the growing demand. Private and public
expenditures for applied research and the development of new useful hydroponics projects have
expanded in many countries. It has a wide monetary effect, giving long haul business, provincial
turn of events, destitution decrease, and really restricting over-double-dealing of local species
(Campo et al., 2018).

Fish, bugs, bivalves and pearls, mollusks, shellfish, and oceanic plants are among the
aquatic organisms that can be reared, and the controlled habitats include ponds, cages, pens, and
(Tunde et al., 2015). Man consumes the majority of these aquatic species to satisfy his need for
high-quality animal protein (Issa et al., 2022). Fish have a crucial role in the economy of several
countries; their contribution to Pakistan's GDP accounts for 1% of the country's overall budget.
They are a good source of food, and cord oil is consumed by almost 1 billion people as an animal
protein(Ali et al. , 2019).Fish is a decent wellspring of creature protein, as well as important
minerals and trace elements. Pakistanranks 34 numbers in the world for fish production (FAO
2016).

Approximately 50% of the fish produced is consumed locally, 22% is exported, and 28%
is utilized in poultry and fish meal(kausar, 2017).It increase intellect and cognitive development
in children, reduced risks of cardiovascular illnesses, and reduced risks of high blood pressure
are all health and nutritional benefits connected with fish eating (Wenaty et al., 2018).The n-3
polyunsaturated unsaturated fats tracked down in oceanic food sources, for example,
eicosapentaenoic corrosive and docosahexaenoic corrosive, are available in not many earthbound
dinner, assume critical parts in cerebrum and eye advancement, as well as the neurological,
metabolic, and immunological frameworks(Fiorella et al., 2021).

Malnutrition and an ever-increasing global population are two severe concerns that
millions of people in developing countries face. To combat these difficulties, fish farming is
projected to play a critical role in supplying nutritious food (Muddassir et al., 2019).Fish
utilization has expanded altogether lately, from 1.0 kg in 1961 to 2.3 kg in 2001, yet it stays low
when contrasted with worldwide midpoints of 9.0-16.3 kg for a similar time span. Fishing is
Pakistan's biggest wellspring of commodity income and assumes a significant part in the
country's monetary turn of events. . Fisheries area give direct work to around 400,000 anglers
and 600,000 individuals in subordinate businesses(Laghari and Studies 2018).

With an 814-kilometer coastline, Pakistan boasts a plethora of fish species that are
hindering the growth of the national and international fish sector (Nazir et al., 2015).Fisheries
development and a supported dependability of fish creation have acquired its expected
importance.Instability in fish creation likewise influences the value steadiness and buyer buying
power(Radhakrishnan et al., 2016).Human disruptions such as climate change, habitat
degradation, biological invasions, and ever-increasing harvesting are putting fish communities in
jeopardy around the world. These acts are to blame for an increase in the rate of biodiversity loss
due to species extinction, as well as drastic alterations in relative abundances and body sizes
among the remaining species (Leitão et., al 2018)).Viruses also lower fish welfare by generating
a variety of situations that negatively impact the fish's well-being, including as reduced feed
intake, aberrant swimming behavior, diseased fish predation, and unfavorable social interactions
(Mugimba et al., 2021).Sickness episodes in the cultivated fishes stayed a significant bottleneck
in the all-out fish creation ( Kibenge et al., 2012).It is assessed that microbes (55%), trailed by
infections (23%), parasites (19%) and organisms (3%) are the most widely recognized reasons
for illnesses in hydroponics (Sahoo et al., 2022).

Pakistan can further develop the fisheries area using current innovation and obligated
assets, as nature gives adequate assets to advance fish sends out in the country(Rehman et al.,
2019). Fishery assets are positive, stocks are lacking and interconnections among them are
profoundly convoluted. For appropriate fishery the board Knowledge of fish stock size,
execution and reaction to fishing pressure is vital(Nazir et al., 2015). Antibiotics are utilized to
treat bacterial infections in aquaculture, and a large number of antibiotics are employed in
aquaculture systems, exerting a strong selection pressure on bacteria that are resistant to
antibiotics (Manage et al., 2018).

As an economically important fish species, the feeding behavior and dietary preference of
fish decide the production costs and profitability of fish aquaculture. Artificial diet feeding of
fish is necessary for large-scale fish reproduction, lowering aquaculture expenses, and increasing
economic efficiency (Shen et al., 2021).The need for fish feed is increasing day by day as a result
of the growth of intensive and semi-intensive systems. Farmers want to save money by using
artificial feed, which is good for the country's economy (Daniel et al., 2016). Fish dinner is one
of the main wellsprings of high-esteem proteins in fish feed today. As a result of its huge
creation volume, dietary substance, and value contrasted with FM, poultry side-effect dinner is
respected a reasonable substitution proteinin artificial diets for carnivorous and omnivorous
aquaculture species (Sabbagh et al., 2019).

Because of its consistent availability, low price, and relatively high protein and balanced
amino acids, soybean meal (SBM) is a viable alternative protein source. It has been shown that
SBM may replace 30–50% of dietary FM in formulated diets ( Liu et al., 2021).Some fish are
higher in fat, while others are greater in protein; in general, fatty fish are sources of lipids with
proven health benefits, while lean fish supply high-quality protein (Mendivil et al., 2021).With
its natural flavor and great digestion, fish muscle tissue is a significant wellspring of imperative
amino acids, fatty acids, vitamins, and mineral components (Ozden et al., 2020).
 One of the most prominent Indian big carp in polyculture is Labeo rohita (rohu). This
species can be found in Southeast Asia, including Sri Lanka, Japan, China, the Philippines, and
Malaysia. It feeds on plankton and is known as a water column feeder. It likes zooplankton in
natural conditions, but green growth and lowered vegetation in the grown-up and adolescent
stages (Majumder et al., 2018). Rohu is one of the most delicious and famous freshwater fish
developed in Bangladesh, India, and different countries nearby, attributable to its extraordinary
development potential and high client interest. It is a decent wellspring of protein and omega-3
PUFA unsaturated fats. It is likewise a wellspring of calcium and nutrient (Girija et al.,
2018).Rohu (Labeo rohita) is a generally developed cultivated fish species in Bangladesh and
other Asian countries like India, Myanmar, Nepal, Pakistan, and Vietnam (FAO, 2018). It
contributes 4% of the significant species created (FAO, 2018). As solitary animal types, it
represents around 15% of the world's freshwater hydroponics (FAO, 2019). Moreover, this
species can undoubtedly be developed with other fish species and performs admirably in
polyculture procedures (Jahan et al., 2021).

The addition of enzymes to aqua feed improves feed consumption. The digestive process
is aided by enzyme supplementation. When enzymes are added to aqua feed, the digestive
system performs better and the feed intake ratio rises. Exogenous enzyme in the meal lowered
anti-nutritional factors while also assisting endogenous enzyme in performing better and
increasing digestion (Gomes et al., 2016).The presence of digestive enzymes and their level of
activity is utilized as a relative indicator of meal acceptance, fish larvae development rate,
digestive capability, and subsequent survival rate(Assan et al., 2022).In the intestinal lumen, the
exocrine pancreas of fish synthesizes and secretes amylases (Bakke et al., 2011). Amylases act in
the intestine and pyloric caecum at an alkaline pH (Nolasco-Soria et al., 2021) Amylase and
protease are key enzymes in plant, animal, and microbial cellular metabolism. Both enzymes
catalyze the breakdown of macromolecules into smaller building components, which are then
used to generate energy or create new biomolecules within the cell. Amylases are enzymes that
break down polysaccharides like starch and glycogen into short-chain sugars. These enzymes are
found all over the body and play an important role in metabolism (Champasri et al., 2021).
Keeping in view all these aspects this project was planned to determine the growth, apparent
nutrient digestibility, digestive enzyme activity and body composition under the impact of
amylase catalyst in Labeo rohita.
Chapter 2

Review of literature:

Ghafarifarsani et al. (2022) concentrated on the utilization of home grown increments and
nutrients to help fish wellbeing stand out. The impacts of L-ascorbic acid (VC), Thymus vulgaris
L. medicinal ointment (TE), and quercetin (QR) supplementation on normal carp improvement,
stomach related catalysts, body organization, and biochemical boundaries are examined in this
review (Cyprinus carpio). 400 and twenty fish weighing 20.46 0.07 g was parted into sets of
three and haphazardly alloted to one of seven exploratory treatments T1 (0, control), T2 (500
mg/kg VC), T3 (1000 mg/kg VC), T4 (1% TE), T5 (2% TE), T6 (200 mg/kg QR), and T7 (800
mg/kg QR) were the fixings in the trial feasts. For 60 days, the fish were taken care of 3% of
their body weight consistently. As per the discoveries, the exploratory eating regimen groups had
a more prominent last weight, weight gain (WG), explicit development rate (SGR), and
endurance rate (SR), as well as a lower feed change proportion (FCR) than the benchmark group
(p.05). Gastrointestinal protease, amylase, and lipase chemicals showed significant upgrades in
undeniably enhanced bunches when contrasted with the control (p.05). At long last, the ongoing
discoveries showed that VC, TE, and QR could effectively increment C. carpio endurance,
development execution, and biochemical markers.

Wiszniewski et al. (2022) led an eight-week try looking at the impacts of taking care of
adolescent divas (Acipenserruthenus) business feed (bunch C) versus exploratory feed improved
with papain at 10 g kg1 feed (P1) and 20 g kg1 feed (P2) (P2). The specialists analyzed neurotic
changes in the liver and stomach, proximal body synthesis, oxidative reaction, immunological
marker articulation, and generally development. Moreover, a Yersinia ruckeri challenge test was
performed. P1 and P2 had essentially higher last body loads (P 0.05) than the benchmark group
(107.07 7.66 g and 111.98 1.93 g, individually) (99.73 2.71 g). The level of digestive not set in
stone by P2 while supranuclear surface still up in the air by P1 and P2. When contrasted with the
other dinner treatment gatherings, Group P2 fish showed discernibly higher - amylase, trypsin,
lipase, and leucine aminopeptidase exercises in their back digestive system (P 0.05).
Champasri et al. (2021) examined the biochemical properties, protein exercises, isoenzyme
design, and sub-atomic load of three kinds of stomach related compound from six freshwater fish
species: Puntiusgonionotus (normal silver spike), Puntioplites proctozysron (Smith's point),
Oreochromis niloticus (Nile tilapia), Hemibagrus spilopterus (yellow mystus),Ompok
bimaculatus (margarine catfish), and Kryptopterus geminus (sheatfish).The ideal pHs for
amylase and basic protease exercises were 7.0-8.0 and 8.0-10.0, individually, and the ideal
temperatures were 45-60 °C and 50-55 °C. Results showed that amylase and soluble protease
from Puntioplites proctozyron had the most elevated action under ideal response conditions.
There were one to three isoforms of amylase and five to eight isoforms of soluble protease in
each fish species, with sub-atomic loads going from 19.5 to 175 kDa. Both basic proteases and
amylases were steady over an extensive variety of pH and temperature.

Barlaya et al. (2021) directed a 60-days development preliminary of rohu, (Labeo rohita),
took care of diets with guar dinner rather than fish feast in 4 m outside concrete tanks with soil
establishment to look at development, feed change effectiveness, cadaver synthesis, and stomach
related protein action. A fish dinner based diet (Control) and diets with fish feast supplanted at
30%, 60%, and 90 percent with guar feast were used as isonitrogenous and isocaloric consumes
less calories. The three medicines had no huge contrasts in development files, feed change and
protein productivity proportion, endurance, or condition factors. While the movement of stomach
related chemicals in the hepatopancreas didn't vary (p > 0.05) across medicines, the action of
stomach related catalysts in the digestive tract was diminished at more significant levels of
guar.Fish from the 90% substitution group had the most dampness and the un-rough protein, fat,
and gross energy levels, as per corpse creation research. Fish with 30% substitution had the most
minimal dampness and the most elevated unrefined protein, fat, and calorie levels. The review
proposed that handled guar feast may be utilized as a fish dinner elective in the eating regimen of
rohu, driving in lower feed costs without forfeiting fish development and quality.

Das et al. (2021)observed the growth of rohu, the production of digestive enzymes, and
natural resistance, the effects of solid-state fermented aqua feed were seen (Labeo rohita).
Methods: Fish feed including sesame oil cake and mahua oil cake was fermented using the yeast
Saccharomyces cerevisiae, and the outcomes were contrasted to a control feed that wasn't
fermented but nevertheless had the same ingredients. Enhancements to fish feed's crude protein
(CP), crude fiber (CF), and anti-nutritional factors (ANF) were made via solid-state fermentation
(p 0.05). After five months of feeding, the group that received fermented feed showed
considerably higher weight increase and nutrient digestibility, including dry matter, unrefined
protein, and ether extricate, and essentially lower feed transformation proportion (p
0.05).Furthermore, we found that fish fed fermented feed had considerably greater levels of non-
specific immunological indicators such lysozyme, myeloperoxidase, and hemolytic activity as
well as intestinal enzyme activity than fish fed control feed (p 0.05). Rohu exhibited better
immune system and development after being fed a food that was fermented with Saccharomyces
cerevisiae.

Abbas et al. (2021)studied how Labeorohita's development, size, and enzyme activity
were affected by fish meals (Rohu). With 240 fish tested, the average weight was 24.772 grams.
With 60 fish in each of the four treatments, there were 120 fish total .The study participants'
body synthesis, development rates, and catalyst exercises were completely examined. Fish took
care of Oryza contrasted fundamentally from fish took care of different types of feed as far as
weight gain, explicit development rate , and feed transformation proportion (P 0.05). T4 put on
more weight than T2 and T3 put on together. The FCR of T4 was lower than that of T1, although
it was still higher than that of T2, T3, and T2. T2 and T3 had substantially higher specific growth
rates (SGRs), while T4 had an average SGR. Crude protein and digestive enzyme activity were
significantly higher when fed Oryza compared to AMG, Aqua, and Supreme.

Yaqub et al. (2021) directed examination to perceive what dietary supplementation with
Bacillus licheniformis meant for the development, hematological, and immunological boundaries
of Oreochromis mossambicus fingerlings. Fish were taken care of at a pace of 2% body weight
with four exploratory eating regimens, including a control diet and three enhanced slims down
containing 105, 107, and 109 CFUg-1, separately, in a 8-week taking care of preliminary.
Development related pointers, as well as hematological and immunological reactions of the fish,
were evaluated at the finish of the taking care of analysis. The feed transformation proportion
(FCR) was decisively decreased, inferring incredible feed use productivity. At long last,
enhancing the food with Bacillus licheniformis can work on the development and soundness of
O. mossambicus by expanding the probiotic content. Immunological reaction was upgraded in
diet bunches with 109 CFUg-1 of probiotic, as estimated by lysozyme action, respiratory burst
action, and phagocytic activity.When contrasted with the benchmark group, this gathering's
movement was impressively higher (P <0.05). At long last, enhancing the eating routine with
Bacillus licheniformis can help O. Mossambicus become quicker and have better wellbeing.

Nottanalan et al. (2021)conducted a taking care of investigation, green pea and

Pisumsativum leaf dinner were taken care of to Labeorohita fingerlings for 60 days (PSLM).
Control (no PSLM per kilogram), LM150 (150 grams for every kilogram), LM300 (300 grams
for each kilogram), CCX (control; 1 gram for every kilogram of cellulase and xylanase, CX
combination), LM150CX (150 grams for every kilogram of PSLM + 1 gram of CX blend), and
LM300CX (300 gram PSLM in addition to 1 gram CX combination). Six trial gatherings of 270
accustomed fish (5.06 0.08 g; loading thickness of 15 fish for every tank) were arbitrarily
chosen, and all fish got similar feast until they were fulfilled. Explicit development rate, protein
proficiency proportion, dry matter absorbability coefficient, amylase action, and muscle aspartate
aminotransferase and alanine aminotransferase exercises were all exhibition measurements
where the LM150CX bunch beat the other exploratory gatherings. The LM300CX and control
bunches both detailed values for these boundaries that were significantly higher (p 0.05) than
those of different gatherings. Hepatic catalase and superoxide dismutase levels were likewise
most reduced in the LM150CX bunch. Compound movement was fundamentally lower (p 0.05)
in the LM300CX and control bunches contrasted with the other dietary gatherings, except for the
LM150CX bunch. IGF-1 articulation in the liver was expanded by the infusion of leaf dinner and
an exogenous chemical. At last, enhancing with 300 g/kg of PSLM and 1 g/kg of CX (1:1) might
be utilized to get a similar impact in the event that the expansion of DORB to the food
meaningfully affects L. rohita capacity to develop.

Sadek et al. (2021) observed the influence of several exogenous enzymes on development
execution in African catfish Clarias gariepinusin 55-day feeding trial. With two amounts of fiber
in the diet, a multi enzyme complex (xylanase, amylase, and protease) was utilized (5 and 9
percent ), Eight isonitrogenous and isoenergetic experimental diets were created, each containing
5.9% fiber without multi enzymes (T1 and T2) and 5.9% fiber with 2, 4, 6% fiber multi enzymes
(T3 and T4). Each of the eight dietary regimens was fed to three groups of Clarias gariepinus
reared in tanks (11.511 m) with 20 fish in each tank. Clarias gariepinus fed enzyme-
supplemented diets demonstrated much higher growth and feed consumption than those fed
standard diets. All enzyme complex groups had considerably higher protein proficiency
proportions, feed transformation proportions, and protein useful qualities. (P 0.05).Exogenous
enzymes had a substantial impact on the whole fish body components (P 0.05); in contrast to T1,
treatments including exogenous enzymes had the highest fat content and a high fiber content (9
percent). In treatments with exogenous enzymes and minimal fiber, blood parameters were also
changed, with liver enzymes decreasing and total protein and white blood cells increasing. In
conclusion, the findings revealed that enzyme supplementation can greatly improve African
catfish Clarias gariepinus growth performance, feed utilization, and health. The group that was
fed a 4% multi enzymes complex with low fiber content (5%) had the greatest outcomes.

Paul et al. (2021) directed a trial on the Indian margarine catfish, Ompok bimaculatus,
which is a highly regarded catfish with a huge continuing in Southeast Asia. An examination was
done to perceive what different food lipid levels meant for hatchlings' development, body
arrangement, and stomach-related and metabolic catalyst exercises. Three isonitrogenous (40%
unrefined protein) counts calories were formed by enhancing fish and vegetable oil (1:1) at 4.5%
(D1), 7% (D2), and 9.5% (D3) levels (containing rough lipid 5.7%, 8.0%, and 10.45%,
separately in consuming fewer calories than D1-D3) to a fish feast and oilcake-based formed
diet. Margarine catfish hatchlings (0.15 ± 0.01 g) in three-fold bunch for a period of 42 days. In
D2 with 8% lipid, the last weight, net weight gain, protein proficiency proportion, and explicit
development rate was impressively (P 0.05) more prominent. All out body dampness and fat
substance were impressively (P< 0.05) more prominent in hatchlings given diet D2. Amylase
action in fish dropped essentially (P 0.05) when dietary fat levels expanded. As per the
discoveries, an eating routine containing 8% rough fat could provide ideal development and
endurance of margarine catfish hatchlings during their initial turn of events.

Olude et al. (2021) decided how changing measures of sundried cassava leaf dinner in the
eating regimens of Labeo rohita fingerlings impacted their development and improvement
throughout the span of 75 days (SCLM). Four isonitrogenous (300 g/kg rough protein) and
isoenergetic (18 MJ/kg) dinners were created by subbing 0, 130, 260, and 390 g/kg of SCLM for
de-oiled rice wheat (DORB). Three separate L. rohita fingerling bunches were arbitrarily allotted
to one of six unique eating regimens (normal weight 2.03 0.03 g). At the finish of the
preliminary, the eating routine 3 gathering and the benchmark group saw comparative rate
weight gains (106.38 4.41%), explicit development rates (1.29 0.04% percent/day), and feed
change proportions (2.38 0.08%) (P> 0.05).p> 0.05). The superoxide dismutase action in the
gatherings given Diets 3 and 4 was fundamentally (p 0.05) expanded contrasted with the Group
Fed Diet 1 (GFD1). In the eating regimens of L. rohita fingerlings, SCLM can supplant up to
166.5 g/kg DORB without compromising development, as per a second-request polynomial
relapse investigation of IGF-I and IGF-BPI articulation.

Gopan et al. (2020) disconnected a protein from karanj seed (KPI), containing 921.2 g
protein/kg seed and a reasonable amino corrosive profile, especially high in methionine. KPI-0
(control, 0 g/kg KPI); KPI-25 (supplanting 250 g/kg SPI protein with KPI); KPI-50 (supplanting
500 g/kg SPI protein with KPI); KPI-75 (supplanting 750 g/kg SPI protein with KPI); and KPI-
100 (supplanting 1,000 g/kg SPI protein with KPI) were figured out for the taking care of
L.rohitaThe KPI took care of and control bunches didn't contrast significantly (p >.05) as far as
weight increment %, explicit development rate, feed transformation proportion, or protein
effectiveness proportion. The control and KPI-25 gatherings had an extensively higher
hepatosomatic record than different gatherings. Aside from ether separate, the entire body
syntheses didn't contrast fundamentally (p >.05) across the gatherings. The activities of stomach-
related (amylase, protease, lipase, and soluble phosphatase) and metabolic catalysts (hexokinase,
transaminases, and lactate dehydrogenase) compounds, as well as glycogen holds, were not
fundamentally different, while gastrointestinal basic phosphatase was altogether unique (p.05).
In the KPI-75 gathering, the RNA-DNA proportion was impressively more noteworthy (p.05).
Thus, the investigation discovered that KPI can totally supplant SPI protein in the eating
regimens of L. rohita fingerlings at a centralization of 191 g/kg.

Debnath & Saikia. ( 2020) examined amylase and protease activity in distinct parts of the
digestive tracts of two teleosts (Rohu, Labeo rohita and Koi, Anabas testudineus), both with
differing feeding patterns (herbivorous versus carnivorous). There were significant changes in
enzyme activity between different parts of the digestive tract. The most amylolytic activity was
found in the posterior digestive tract with three equal portions of the stomachless gut, but the
highest proteolytic activity was found in the mid area. Despite this, the Koi pyloric caeca, which
has a three-part digestive system that consists of the stomach, pyloric caeca, and intestine, had
the highest specific activity for amylase and total protease. At a pH of 8.0, Rohu's frontal and
median DT showed the highest amylase activity. On the other hand, the last stage of the DT
showed that a pH of 7.0 is ideal for amylolytic activity. The ideal temperature for amylase
activity was 35 °C throughout the DT. It was found that amylase and protease activity require a
specific pH and temperature.

Goswami et al. (2020)examined how plant-based diets affected the digestive system of
Labeorohita fingerlings. The control diet consisted of fishmeal as the only source of protein (F).
Four test meals, each providing 300 g/kg protein, were made from combinations of water fern
Salvaniamolesta (FSM), duckweed Lemna minor (FLM), and almond oil cake Terminalia
catappa (FTC) (FTCLMSM). The outcome of this experiment showed that the fish's digestive
enzyme activity was impacted by the diet composition. As a result, as compared to rohu eaten the
other meals, those fed FLM showed significantly higher levels of amylase, trypsin, and protease
activity.

Sangavi et al. (2020)directed a multi-day taking care of preliminary to test the usage
probability and nutrional worth of palm part feast for juvenile Labeo rohita. Palm kernel meal
was used to substitute sunflower oil cake and rice brans in five different iso-nitrogenous diets,
namely control, T1, T2, T3, and T4. Five iso-nitrogenous diets were prepared with various
inclusions of PKM which replaced sunflower oil cake and de-oiled rice bran namely, control
(without PKM), T1 (50 g/kg PKM), T2 (100 g/kg PKM), T3 (150 g/kg PKM) and T4 (200 g/kg
PKM). The growth performance values showed a higher overall linear trend in T2 and it is
significantly different from control as well as T1 groups. Results revealed that the maximum
body weight, protein efficiency, growth rate, and protein utilization were found to be dose-
dependent. T2 has increased activity of amylase and protease.

Zishan et al. (2019) tested the use of sweet potatoes leaf feast as a trade for de-oiled rice
grain in the eating regimens of rohu (Labeo rohita) fingerlings more than a 60-day time frame.
SPLM has a high protein content (22.12%) as well as edible energy (11.81 MJ kg-1). Five iso-
nitrogenous (30%) and iso-caloric (13.5 MJ kg-1) abstains from food were ready by subbing yam
leaf dinner for DORB at 0% (C), 25%, 50% , 75% , and 100%. Each taking care of treatment was
assessed multiple times, with 12 fingerlings for every tank.In a properly randomized manner.
Except for amylase activity, which increased dramatically in the 50 percent SPLM replacement
group, the digestive enzyme activity was unaltered. In the therapy groups, chymotrypsin levels
dropped significantly (p< 0.05).

Sajina et al. (2019) observed how fucoidan-rich seaweed extract (FRSE—20 g kg1) and
exogenous -amylase (100 mg kg1) affected Labeo rohita growth response, metabolic enzymes,
immunological markers, and amylase gene expression. Four pure iso-nitrogenous diets (350 g CP
kg1 feed) was made with different -amylase combinations. The -amylase fed groups had
considerably higher intestinal amylase activity, while the -amylase supplemented groups had
significantly reduced amylase gene mRNA expression (p 0.05). As a result, supplementing the
food of L. rohita fingerlings with 100 mg /kg -amylase and 20 g /kg FRSE has a growth-
promoting impact without impairing the immune-modulating effect.

Patil et al. (2019) studied exogenous enzymes such as cellulase, hemicellulose, and
alpha-amylase were added to the diets of catla fry at concentrations of 0.5, 1, and 1.5 percent,
respectively. For a period of 90 days, the experiment was conducted in circular plastic containers
with a capacity of 125 L, with twelve treatments and two repetitions. From an initial length range
of 17 – 19 mm and a weight range of 0.37 – 0.45 g, the catla fry were raised to 35.8 – 47.9 mm
in length and 5.53 –4.84 g in weight. The therapy incorporating a mixture of exogenous enzymes
each at 0.5 g 100 g-1 level in the catla fry diet resulted in the highest length, weight gain, specific
growth rate, and survival.

Maiti et al. (2019) led a 60-day taking care of preliminary to decide the most ideal way to
take care of Hygrophila spinose leaf dinner (HSLM) to Labeo rohita (rohu) fingerlings as a trade
for de-oiled rice grain (DORB). LM10 (10% HSLM), LM20 (20% HSLM), LM30 (30%
HSLM), CEE (control + EE), LM10EE (LM10 + EE), LM20EE (LM20 + EE), and LM30EE
(LM30 + EE) were the eight isonitrogenous (31 percent unrefined protein) and isocaloric (330
Kcal DE/100 g) exploratory eating regimens with or in sets of three, 288 fingerlings were
haphazardly scattered in eight trial gatherings. All through the trial period, fish were taken care
of to satiation with the proper exploratory eating regimen. In contrast with control, fish taking
care of a 10% HSLM-containing diet showed impressively (p 0.05) higher weight increment
percent, explicit development rate, RNA content, and RNA-DNA proportion. The 20% and 30%
HSLM took care of gatherings were demonstrated to be non-huge (p > 0.05). Exogenous
compound supplementation in leaf dinner based eats less (LM10EE, LM20EE, and LM30EE)
didn't essentially increment fish development execution when contrasted with non-enhanced
partners (LM10, LM20, and LM30). K

Iqbal et al. (2018) thought about the impact of fishmeal and chosen plant-beginning feed
fixings on stomach-related catalysts' action as well as on the histology of the liver and digestive
system in Labeo rohita. The four treatments slimmed down included guar feast (GM), soybean
dinner (SBM), cotton seed feast (CSM), and canola feast (CM) as trial feed fixings, with a
fishmeal (FM) contained diet filling in as the control. Every one of the treatment and control
slims down had three imitates. The trial was completed in 15 fiber-glass aquariums containing
ten fish each. Each tank's fish were taken care of at 4% of their body weight. The consequences
of the preliminary showed that protease action contrasted impressively (p 0.05) between the
front and back pieces of the digestive system, but the activities of amylase and lipase in the
complete digestive system varied fundamentally (p 0.05) between the medicines.

Kumar et al. (2018) noticed the wholesome potential segregated from elastic seed in the diet of
Labeo rohita. Five isonitrogenous and isocaloric exploratory eating regimens were created with
shifting degrees of RPI instead of soybean protein seclusion (SPI), for example, 0%, 25%, half,
75%, and 100 percent, and were named control, RPI25, RPI50, RPI75, and RPI100, separately.
The all-out body creations and stomach related/metabolic catalyst actions of the various
gatherings were not considerably unique (p>0.05). While looking at the control and RPI groups,
blood cholesterol and fatty oil levels were viewed as impressively higher (p 0.05) in the
benchmark group. Generally speaking, this study shows that RPI derived from elastic seed can
be utilized as an elective protein source in the eating regimen of rohu fingerlings without
influencing improvement, supplement usage, or physio-metabolic reactions.

Ranjan et al. (2018) concentrated on the supplement utilization and development

execution of Labeo rohita. They took care of T1 [DORB enhanced with phytase and xylanase
(0.01 percent each)] for 60 days. T2 [T1+L-lysine (1.4%), L-methionine (0.4%), mix of EPA and
DHA (0.5%)], T3 [DORB enhanced with phytase (0.01), blend of xylanase and cellulase
(0.075%), L-lysine (1.4%), L-methionine (0.4%), mix of EPA and DHA (0.5%)], T4 [DORB
enhanced (monetarily accessible carp feed). T4 bunches had the most elevated rough protein
edibility. The T2, T3, and T4 bunches showed comparative stomach-related compound exercises,
though the T1 bunch had the least action of these catalysts. On account of the better in vivo
edibility of T3 feed (unrefined protein 18.18%) contrasted with T4 diet (rough protein 32.01%),
it is presumed that T3 feed (unrefined protein 18.18%) has similar development execution to
Labeo rohita.

Phulia et al. ( 2018) determined the nutritional potential of fermented Jatropha kernel
meal in diet of rohu. A 60-days experimental trial was carried out. Control, T1, T2, and T3 are
four iso-nitrogenous diets containing JKM that were created. Hepatosomatic index, amylase, and
catalase activities did not differ substantially between diet groups. Results showed that fish had a
higher total body composition at the end of the study. As a result, rohu fingerlings effectively use
FJKM without compromising their growth.

Gajender et al. (2018) conducted an experiment to study the enzymatic profiles of three
major Indian carps in managed and unmanaged polyculture systems in Haryana, India. Catla
(Catla calta), rohu (Labeo rohita), and mrigala were the three species (Cirrhinus mrigala).
Phytoplankton was found to be considerably (p <0.05) dominant in the gut contents of C. mrigala
from both ponds. The gut of L. rohita exhibited equivalent levels of phytoplankton and
zooplankton, whereas the gut of C. catla was heavily dominated by zooplanktons. C. mrigala
had increased specific cellulase and amylase activity. It was also discovered that these behaviors
are more prevalent in managed ponds than in unmanaged ponds. The presence of higher levels of
protease and amylase activity in L. rohita corroborated the fish's omniplanktivorous nature. In
compared to other enzymes, digestive enzymes from the gut of C. mrigala indicated higher
lipase, cellulase, and amylase. C. mrigala was phytoplanktivorous, L. rohita was
omniplanktivorous, and C. catla was zooplanktivorous, according to the findings. Fish raised in
regulated ponds appeared to have more enzyme activity in their guts, implying that they would
grow faster. The study's findings add to our knowledge of feeding patterns in polyculture
systems at various stratus levels within the available nutrients.

Khalil et al. (2018) investigated the influence of exogenous enzyme (-amylase) added
feed on growth performance, health, and hematological analysis of Pangasianodon
hypophthalmus (starting average weight 32.460.36 g) in a 90-day feeding trial in glass aquaria.
T1, T2, T3, and T4 were created by combining feed components with 0.75, 0.50, 0.25, and 0.00
g kg-1 of -amylase as T1, T2, T3, and T4 respectively (control). At the conclusion of the feeding
study, a 100% survival rate was recorded. In comparison to the control group, fish fed enzyme
enhanced diets grew at a much faster rate. Treatment # 2 showed the greatest increase in growth
rate. Treatment #3 (0.75g kg-1 -amylase) had the highest protein content of 68.18 percent. All
enzyme supplemented groups had higher specific growth rate (SGR), feed conversion ratio
(FCR), and condition factor (CF) than the control group (p0.05). The findings revealed that
adding enzymes to the diet helped P. hypophthalmus develop and stay healthy.

Bishnoi et al. (2017) investigated the effect of aloe Vera as a fish food supplement on
Labeo rohita growth and metabolism. For this aim, six graded levels of Aloe Vera pulp 0(D1),
100(D2), 200(D3), 400(D4), 600(D5), and 800(D6) g/kg) were combined in a basal diet and fed
to experimental fish (at 4% of body weight each day) for a total of 60 days. Aloe Vera
supplementation had a considerable effect on fish growth. D4 had the highest weight gain
(60.4100.996g), percent weight gain (275.70014.683%), SGR (2.2030.066%), and GCE
(0.5790.011) values. When compared to other treatments, the fish fed D4 showed greater food
use and a lower food conservation ratio (FCR) of 1.7260.032. Similarly, Amylase activity
(27.4800.068), Protease (28.4560.255), and Lipase (0.63330.015) also had greater values in D4.
As a result, it can be stated that Aloe Vera @400g/kg diet improved rohu development and
metabolism significantly.

Murtaza et al. (2016)noticed the movement of stomach related compounds in

Hypophthalmichthys molitrix, Catla catla, and Cyprinuscarpiofed with various feed fixings.
Each species was represented by two glass aquaria (322 ft) with ten fish in each. For 70 days,
fish in one aquarium were fed rice polish while those in the other tank were fed corn gluten at
3% of their wet biomass. The moist weight of the fish differed significantly between those fed
rice polish and those fed corn gluten, according to the findings. Those fed corn gluten grew
considerably faster than fish fed rice polish. Amylase and lipase activity were greater in all fish
species given corn gluten. The liver had higher amylase activity than the intestine, but the
intestine had higher lipase activity. It was discovered that different fish species react to different
feed elements in different ways.
 Ojha et al. (2016) performed experiment to check the effect of herb supplemented diet on
growth, metabolism and Haemato-immunological parameters of Indian Major Carp, Labeo
rohitafingerlings.The fish were fed a food containing four different levels of Mucuna pruriens
and Pedalium murex (0.0, 0.6, 0.08, and 0.1 g/100g diet) (1; 1). The outcome of improved
growth at treatment level 0.06 g/100 was supported by a rise in digestive enzymes such as
protease, amylase, and lipase. However, when compared to the control, the enzyme involved in
protein metabolism was significantly boosted. The findings revealed a significant impact of an
herbal combination of Mucuna pruriens and pedalium murex on Labeo rohita fingerling growth,
metabolism, and immune defence mechanisms.

Umalatha et al. (2016) concentrated on the varieties in the exercises of a few stomach-
related catalysts in various size groups of Indian significant carp, Labeo rohita. The
appropriation of stomach-related proteins in four digestive fragments of grown-up fish was
likewise explored. Amylase, trypsin, complete protease, and chymotrypsin action ascend with
the length and weight of the fish. In contrast with different pieces of the digestive tract, the
proximal digestive tract had the most elevated amylase activity. The distal digestive tract had the
most extreme cellulase action, while the hepatopancreas had none.

Adeoye et al. (2016) directed review to research the joined impact of exogenous chemicals and
probiotic supplementation on tilapia improvement, digestive morphology, and microbiome
arrangement. Tilapia (34.56 ±0.05 g) were taken care of one of four eating regimens (35%
protein, 5% lipid); one was a control, and the other three were enhanced with compounds
(phytase, protease, and xylanase), probiotic (containing Bacillus subtilis, Bacillus licheniformis,
and Bacillus pumilus), or enz-expert (containing Bacillus subtilis, Bacillus (the blend of the
chemicals and probiotic). As far as conclusive body weight (FBW), explicit development rate
(SGR), feed transformation proportion (FCR), and protein effectiveness proportion, tilapia took
care of the enz-master diet beat tilapia took care of the control and probiotic enhanced eats less
(P 0.05). (PER). To sum up, enhancing the dinner with a mix of proteins and probiotics can
further develop tilapia improvement and digestive morphology while adversely affecting the

microbial sythesis of the stomach.

Chapter 3

Materials and Methods

This study was intended to notice the impact of exogenous enzyme amylase supplementation on
growth, apparent nutrients digestibility, digestive enzyme activity and body composition of
Labeo rohita. The course was held in the Research Lab, Department of Zoology, and
Government College University Faisalabad and went on for 90 days.

3.1 Experimental fish

Labeo rohita fingerlings were brought from Punjab Fish Hatchery, Santayana Street, Faisalabad.
Prior to beginning, 90 days of preliminary fingerlings were accustomed to for several weeks and
fed with a control diet (crude protein 32%) at the pace of 4% of wet body weight (Rowland and
Ingram, 1991). After acclimatization, 150 fingerlings were secluded in six tanks each with a
recreated aspect (90 cmL30 cmW45 cmH) with a 30 L water limit. Broken up oxygen was
observed on a regular basis by utilizing an oxygen siphon with a slim framework.

3.2 Feed ingredients and feed preparation

Feed ingredients used for this experiment was soybean meal, corn flour, fish meal soy bean oil,
wheat flour and vitamin premixes. All the ingredients were purchased from the local market and
finely grounded in lab. To prepare the experimental diet amylase (3mg and 6mg) with the
activity unit of 1300 and all ingredients were mix together in an electrical mixer for 10 mints.
After this fish oil was added and continued mixing for 5 minutes (Lovell, 1989). 15 -20% water
was added to prepare the dough then cut into pellets using pelletize machine and dried in oven at
105oC for 16 hours. After drying, feed was stored into air tight jars.Two experimental and one
control diet was prepared. The diet was formulated by Linear Formulation method using win
feed 2.6(UK).

Experimental Diet

3.3 Feeding Protocol/ Experimental design:

After acclimatization, every one of the fingerlings of uniform size (2–3 g) was chosen
haphazardly, weighed by utilizing electronic equilibrium, and circulated into six fish tanks. Each
tank had 20 fingerlings of nearly equal size scattered with no obvious end goal in mind. The
examination was led as a Complete Randomized Design (CRD) with one control and two
medicines, viz., T1: amylase 3mg/kg, T2: amylase 6mg of fish feed. Feeding was allowed once
per day at 4% of live wet body weight. The water quality boundary, for example pH, break up
oxygen, carbon dioxide, was checked through YSI expert series multi-boundary proficient in
addition to the meter. Broken up oxygen (5 ppm) was kept up with via pneumatic machines box
and slim framework (APHA 1998). During the preliminary, based on fortnightly control and
treatments, the live wet body weight and body length of 15 fingerlings were recorded. The body
length of fingerlings was estimated by utilizing a 30 cm ruler from the tip of the mouth to the tip
of the tail balance. Body weight was estimated by utilizing logical equilibrium. Following 2
hours of taking care of the meeting, the waste material was gathered. On a consistent schedule at
60 oC, the waste material was dried and afterward put away in the containers for additional
examination (Allan and Rowland, 1992).

3.4 Growth study:

Fingerlings growth was calculated according to the method followed by (Brown, 1957).

Expansion of net body weight (g):

Normal body weight of first noticed fortnight-Average body weight of next noticed fortnight.

Expansion in net all-out length (cm):

Normal body length of first noticed fortnight-Average body length of next noticed fortnight.

Condition factor:

K = W × 105÷L3

Where,

W=Average wet body weight (g)

L=Average body length (cm)

(Htun Han, 1978)

Specific growth rate

SGR=In (Final wet body weight) - In (initial wet body weight) × 100

Time duration (Days) × 100

Weight gain = Final weight – Initial weight

Weight gain% = Final weight – Initial weight ×100

Initial weight

Protein efficiency ratio (PER):

PER= Final body weight - Initial body weigh

Amount of protein fed

3.5 Logical strategies

An example of a gathering of feed, stove dried dung, and body meat were analyzed for dry
matter (DM) utilizing broiler vanishing at 105°C, crude protein (CP) utilizing microkjeldahl
examinations, and gross energy utilizing an oxygen bomb calorimeter, all in accordance with
AOAC (1995) conventions. The 10454 Soxtec framework HTz and the chloroform/methanol
extraction procedure were utilized to ascertain the oil content (Blight and Dyer, 1959).

3.5.1 Dry matter (DM)

Samples of1 gram body meat, fecal and feed were oven dried to determine the dry matter of
sample. Dry matter was the residue left behind after drying.
Procedure

One gram of body meat, feed, and feces were set in a petri dish. Weight this petri dish. This is
w1. Set this petri dish on the stove at 105 oC for 16 hours. Then the example was moved to the
desiccator for 5 minutes. The dried example is then weighted, which is w2. The decline in weight
was determined as moisture, from which the dry matter was determined by utilizing the recipe:

Dry matter=100−moisture%

W1 - W2
Moisture%= × Wt. of sample 100

Where:

W1= weight of petri dish+ test prior to drying

W2=weight of petri dish+ test prior to drying

3.5.2 Ash

Electric furnace was used for ash analysis. Ash is the measure of inorganic content in the sample.

Procedure

For ash analysis muffle furnace is used. The temperature of furnace is set at 500 to 600C. Before
ashing the weight of sample is W1 and after ashing the weight of sample isW2.Volatile
components and water evaporates, in the presence of oxygen in the air organic substance burned
and change into N2, CO2 and H2O.

Ash= (w1- w2) / (weight of sample) × 100

 3.5.3 Crude protein analysis:

Principal

Micro kjeldahl apparatus was used to evaluate the crude protien of fecal matter, body meat
and feed.To assimilate the sample, acid was used, N 2is predicted by titration. Crude protien
was calculated from the amount of nitrogen in sample.

Procedure

Three main steps of this analysis were digestion, neutralization and titration.

Digestion

Potassium and copper sulfate were combined in a 93:7 ratio to create the digesting solution.
30 cc of concentrated H2SO4 were added to a kjeldahl flask along with 1 gram of sample and
5 grams of the digestion mixture. On the sizzling plate, the last drop of the kjeldahl flask had
disappeared. The heat was gradually increased after the mixture had first been cooked at a
low temperature. Until the liquid turned greenish blue, the procedure was repeated. After
consuming food and liquid for three hours, the digestive process was finished. When the
liquid had cooled to room temperature, distilled water was added to thin it out.

N (food) (NH4)2SO4

Neutralization

After being cooked, micro kjeldahl was washed with water. The apparatus was then loaded
with 10 ml of 40% NaoH and subjected to vapor pressure distillation.

(NH4)2SO4 + 2NaoH 2NH3 + Na2SO4

In order to collect ammonia, a 10% boric acid solution was prepared, added a drop of methyl
red and, afterward, positioned it underneath the contraption's end pipe.

NH3 + H3BO3 (boric acid)→2NH4+ +H2BO3-(borate particle)

It took a continual stream of ammonia collection to turn the pink solution into golden yellow.
The amount of 0.1 N H2SO4 needed to neutralize the ammonia in the boric acid was
determined by a titration. Both the nitrogen used to color-change the indicator and the
nitrogen added to the solution were the same amount.
 H2BO3- +H+ H3BO3

The recorded value of the crude protien was assessed by utilizing following formula:

N2 (%) = Volume of 0.1 N (H 2SO4 ) used x 0.001 x 250 x 100

Weight of sample x 10

Crude protein percentage = %N2×6.25

Where:

0.001= volume of a 0.1 N H2SO4 used to kill 1ml of smelling salts

250ml = weakening of processed blend

100 = level of nitrogen

10 = volume of weakened and processed example

6.25 accepted factor for condition of N2% to rough protein

3.5.4 Crude fat extraction:

Unrefined fat was investigated by utilizing soxtec HTz 10454 contraption.

Principal

Diethyl ether can be used to remove lipids, oils, and fats from a dried sample prior to the
ether being evaporated to leave only the dry component. Weighing the remaining raw fat
or ether extraction comes next. Prior to drying, the perspective sample and ether must be
cleaned to prevent extraction of water-soluble components (glycerol, lactic acid, glucose,
and urea). The leftover moisture in the sample was dried at a lower temperature to
prevent the lipid from being damaged.
 Procedure
On the waste, food, and body meat of a Soxtec adopter, one gram of defatted cotton wool
was placed. After that, we poured wood into the condenser and lowered the heating plate
holder. Amounts of up to 50 to 70 ml of petroleum were weighed into the extraction cup
before turning on the machinery with the cold tab. For around 30 minutes of the
extraction, the mode knob was in the boiling position. Using the thimble, the unlocked
condenser valve was switched to the washing position, where the boiling mixture was
held for approximately 10 minutes. We released the charge in the extraction cup, opened
the condenser valve, and turned off the master switch. Later, cold water was added. Lard
and petroleum ether were heated in an extraction cup. The extraction cup was weighed
once more after drying, and then it was placed in a desiccator for five minutes.
Fat percentage was determined by utilizing the following structure:

Percentage of crude fat = W2-W1 ×100

Wt. of test

3.5.5 Nitrogen free extracts (NFE %)

By taking away the amount of % fat, protein, and dampness from 100, the nitrogen free
concentrates were determined.

Nitrogen free concentrate % = 100-(protein+ dampness +fat)

1. Gross energy (Kcal g-1)

Feces energy parts of feed and body meat was determined by utilizing following equation:

GE (Kcal| g) = (5.64× protien %) + (9.44× lipid %) + (4.11× NFE %)

3.6 Approximate nutrients utilization

By using following formula approximate nutrients utilization of feces and feed

were calculated:

Approximate nutrients utilization = Dry matter in feed-dry matter in feces × 100

Dry matter in feed

3.7Apparent digestibility coefficient equation
By using following equation apparent digestibility coefficient (%) of each experimental diet
was calculated (Maynard, 1969)

ADC of dry matter (%) = 100 × [dietary Cr 2 O3| fecal Cr2O3) × (fecal nutrient or energy
concentration | dietary nutrient or energy concentration)

Apparent digestibility equations:

Apparent digestibility of supplements for trial eating routine was computed by utilizing the
standard technique Maynard and Loosli (1969) and the apparent digestibility coefficient for test
fixing by Sugiura et al., (1998).

Nutrient of Diet (%) = 100 × [1-%marker in diet/% marker in feces × % nutrient in feces
/%nutrient in diet]

 
ADC I = ADCT +  1- S  D R sD1  x ADC T - ADC R 

Where:

ADCI =Apparent digestibility coefficient of test ingredient;

ADCT=Apparent digestibility coefficient of test diets;

ADCR=Apparent digestibility coefficient of the reference diet;

DR=%nutrient of the reference diet;

DI=%nutrient of the test diet;

S=Proportion of test ingredient in test diet;

1-S=Proportion of reference diet in test diet;

The FCR for every treatment will be processed by the accompanying condition.

FCR=F/(Wf - WO), where

F is the heaviness of food provided to fish during the review time frame,

WO is the live weight of fish toward the start of the review time frame,

Wf is the live weight of fish toward the finish of the review time frame.
3.8 Enzyme activity Assays (U mg-1)
Enzyme activity assay is the laboratory method used to measure the enzymatic activity. This is
the measure of quantity of active enzyme present and dependent on the condition which is
specific.

3.8.1 Procedure for sample preparation

Three fishes were taken from each tanks of treatment after completion of experiment. The fish
was anaesthetized with the clove oil and analyzed to gather the stomach and liver for the
estimation of enzyme assays. Gut and liver was homogenized in distilled water at 4C for the
preparation of supernatants. Then homogenate was centrifuge at 500rmp for 20 minutes.
Supernatants were stored at -20C until required. The activity of amylase was measured by using
starch hydrolysis according to the Somogy- Nelson colorimetric method.

3.8.2 Amylase enzyme Assay

Preparation of DNS Reagent

Took 10ml of distilled water in a beaker and 500mg DNS was added in it with continuous
stirring. Then 15g of sodium potassium tartrant was added slowly with continuous stirring. After
this added 2M NaoH until solution become clear.

Preparation of starch solution:

By adding 1g of starch in 100ml 0f distilled water starch solution was prepared.

Sample testing:

Two ependrof were taken and 200µl of starch solution was added in each. Then 200µl of
distilled water was added in one and 200µl sample was added in other and put them in incubator
for 3 minutes and 400µl of DNS is added in each ,for 5 minutes boiled in water bath .After this
cool them and added 4 ml of water. Checked the absorbance and put value in formula.

Enzyme activity (U/ml)= absorbance of enzyme – absorbance of blank

Incubation time × dilution factor (Hidalgo et al.1998)

3.9 Chromic oxide analysis:

It all began with a 50 mg dry feed or feces sample containing chromic oxide that was wrapped in
filter paper and put in a micro kjeldahl flask. Nitric acid (5 ml) was added, and it was left alone
for 5 minutes. A yellowish tinge was seen after heating the sample for 20 minutes. Afterward,
pour distilled water into a 100-milliliter volumetric flask halfway, and allow it to cool to ambient
temperature. After 5 minutes, we blended and put subsamples of the solution in calorimetric
tubes, and then we used a U-2001 US-VIS spectrophotometer to assess the 350n wavelength.
The chromic oxide content was estimated by utilizing an economically accessible alignment
bend. The cycle was rehashed, but this time refined water was utilized as the clear and
unadulterated chromic oxide filled in as the norm. For the standard bends at 2, 8, 14, 20, 26, 32,
38, and 44 ppm, further weakenings of the chromic oxide standard were made utilizing the
accompanying recipe:

M1V1= M2V2

Where,
M1= Molarity of concentrated solution
V1= Volume of concentrated solutions
M2= Molarity of diluted solution
V2= Volume of diluted solution
3.10 Statistical Analysis:
After finding the results on growth, apparent nutrient digestibility, digestive enzyme activity and
body composition all data was subjected to statistically analysis by utilizing SPSS software
(Steel et al., 1996).

Chapter 4

RESULTS& DISCUSSION

This study was conducted to determine the effect of enzyme supplementation (Amylase) on
growth, apparent nutrients digestibility, digestive enzyme activity and body composition ofLabeo
rohita .The study lasted for90 days. One control and two experimental diets were given twice a
day. During the experiment, growth parameters weight and length was recorded on fortnight
basis. Fecal matter was collected daily to evaluate the apparent nutrients digestibility. At the end
of the trial gut and liver was collected from the fish to evaluate the enzyme activity. Results of
the subjected data analyzed by using SPSS have been given below:

4.1 Study of growth parameters (body weight, body length, condition factor, specific growth rate,
protien efficiency ratio)

4.2 Analyzed percentage (%) of nutrients in feed (moisture, dry matter, ash, crude protein, crude
lipid, gross energy)

4.3 Analyzed percentage (%) nutrients in fecal matter

4.4 Approximate nutrient utilization

4.5 Apparent digestibility coefficient (%)

4.6 Proximate nutrients percentage (%) in body meat

4.7 Digestive enzyme activity assay

4.1 Study of growth parameter

The basic growth parameters length and weight of Labeo rohita is recorded on fortnight basis to
evaluate the growth. The results were discussed under following parameters:

Body length

Body weight

Body weight

Weight gain was measured by subtracting the previous weight from the current one. Weight was
measured in grams and on fortnight basis.

Fish fed with the experimental diet AM1 (amylase: 3mg/kg) showed the highest increase in body
weight 4.8g with the initial weight of 2.3g. Maximum weight was shown at 2 nd fortnight
(October) 0.55g while the lowest weight gain at 5 th fortnight (Dec) 0.1g.Total increase in weight
in this group was 0.41g.In experimental group AM2 (amylase: 6mg/kg) final body weight was
recorded 4.75g with the initial body weight 2.4g. Maximum weight gain was shown at the 2 nd
fortnight (Nov) 0.75g however the lowest weight gain was shown at the 3 rd fortnight (Nov)
0.3g.Total increase in weight was 0.38g.In control group the final average body weight was
observed 4.8g with the initial body weight of 2.35g. Maximum weight gain is shown at 2 nd
fortnight (October) was 1.15g while the lowest weight gain 0.1g was at 4th fortnight (Dec) .The
total weight gain of this group was 0.41g. (Appendix # 2)

When data was analyzed statistically analysis of variance showed the highly significant

(p<0.01)variations among the fortnights whereas significant (p<0.05) variations were

Observed within treatmentswhile there was non- significant (p>0.05) variations observed

When fortnight and treatments interactionwas made.Turkey’s test was used to find out

analysis of variance (Table 4.1)

When data was analyzed statistically comparison of mean within the treatments showed that
treatment AM1 showed highly significant variations from control group and AM2 .while
treatment group AM2 and control group showed non-significant variations. When for fortnights
comparison of means was observed fortnight F6 (JAN) showed highly significant variations from
F3, F4and F5 (NOV, DEC, DEC, respectively).Similarly, F1 (OCT) and F2 (NOV) fortnights
follow the same trend but showed variations from other fortnights (Table 4.2)

Graphical representation showed the maximum weight gain at 2 nd fortnight (NOV) and lowest
at1st fortnight (OCT) in control group.Whereas among treatments minimum increase in body
weight was observed in AM1 (Amylase 3mg/kg). While in treatment AM2 the highest average
body weight was observed at 2nd fortnight (NOV) (Figure4.1).

The line chart graphical representation showed that maximum increase was observed in control group at
2nd fortnight (NOV whereas minimum gain was at 4 th fortnight (DEC).While among treatments AM1
(Amylase 3mg/kg) maximum increase in body weight was observed at 6th fortnight (JAN) i.e.0.55g and
minimum value showed at 5th fortnight . Treatment AM2 showed maximum increase in body weight at
2nd fortnight (NOV) while minimum at 4th fortnight (JAN). While minimum increase in body weight was
observed in AM1 (Fig 4.2)

Body length

The body length of fish is measured from tip of snout to the end of large lobe of tail fin.With the
help of scale increase in length is measured after every fortnight. In experimental group AM1
(amylase: 3mg/kg) shows the highest final body length 7.9cm with initial body length of
5.3cm.Maximum length gain was observed at 6 th fortnight (Jan) 0.75cm however lowest in the
first fortnight (October) 0.3cm.The total increase in length of AM1 was 1.97cm.In AM2 fish fed
on experimental diet (Amylase: 6mg/kg) shows the final length of 8.75cm with the initial body
length of 5.25cm. Highest increase in body length was observed in 2nd fortnight (Nov) 1.05cm
while lowest was at fourth fortnight (Dec) 0.17cm. Total increase in length was 3.23cm.In the
control group final body length gain was observed 9.55cm with initial body length of
5.4cm .Maximum increment in length was observed at 2nd fort night(Nov) 1.65cm while lowest
was at 1st and forth fortnight (October, Dec) 0.25cm respectively. Total increase in length was
0.69cm (Appendix 4).

When data was analyzed statistically analysis of variance showed highly significant

(P<0.01)variations within fortnight and treatments. When fortnight and treatments

Interaction was made there was non-significant (P>0.05) variations were observed (Table4.3)

When data was analyzed statistically comparison of means of body length indicated that between
the treatments AM1 (Amylase 3mg/kg) showed highly significant variations from other
treatment groups while control group and AM2 (Amylase 6mg/kg) showed non-significant
variations. Comparison of mean between the fortnights showed that F6 was highly significant
from other fortnights while fortnight 3rd, 4th and 5th showed non- significant variations among
each other. While fortnight 1 and 2 ndshowed non- significant variations among each other but
showed highly significant variations from other fortnights (Table 4.4)

Graphical representation showed the overall maximum increase in body length in

control group shows maximum peak at 2nd fortnight (NOV) and lowest peak at 1 st fortnight
(OCT).In treatment group AM2 (Amylase 6mg/kg) maximum increase in body length was
recorded at 2nd fortnight (NOV) while lowest was observed at 4 th fortnight. Whereas in treatment
group AM1 lowest peak was observed at 1 st fortnight whereas the highest peak was recorded at
6th fortnight (Figure 4.3)

Line chart graphical representation showed the peak value at 2 nd fortnight (NOV) and the lowest
was observed at 4th fortnight (DEC). Whereas for treatment group AM1 maximum value
(0.75cm) was recorded at 6th fortnight(JAN) while the lowest value (0.3cm ) was observed at
5th fortnight(JAN).While for treatment group AM2 highest peak was observed at 1 st
fortnight(OCT) and lowest was recorded at 5th fortnight (Figure 4.4).

Condition factor

Condition factor is a numerical value given to a fish that reflects its condition. This value
is arrived at by using a mathematical formula that takes into account both the weight and length
of the fish. It is the study of health status of fish at different fortnights by calculating length to
weight ratio. A well-conditioned fish has a high condition factor, while one in poor condition has
a low factor. It helps us to understand and assess the weight gain along with increase in length.

The highest value of condition factor in AM1 (Amylase: 3mg/kg) was 1.44 at 2 nd fortnight
(NOV) while lowest value was 0.9 at 5 th fortnight (DEC) .The lowest value in control group was
observed at 6th fortnight (Jan) 0.55 and higher value was recorded 1.35at 1 st fortnight
(OCT).Whereas the higher value of condition factor was observed 1.09 in AM2 (Amylase:
6mg/kg) at first fortnight (October) and the lowest was 0.70 observed at 5 th fortnight (DEC).
(Table 4.5)

When data was analyzed statistically analysis of variance indicated highly significant
(P<0.01) variations was observed within the fortnights and within the treatment and also when
interaction between treatments and fortnights was made (Table 4.6).
When data was analyzed statistically comparison of mean of condition factor AM1 (Amylase
3mg/kg) showed highly significant (P<0.01) variations between the treatments as compared to
control group and AM2.Whereas control group and AM2 showed non-significant (P>0.05)
variations. Between fortnights F1 (OCT) and F2 (NOV) highly significant (P<0.01) variation
was observed from other fortnights, while F3, F4, F5 and F6 showed non-significant (P>0.05)
variations from each other. In control group F1, F2 F5 & F6 respectively showed highly
significant variations (P<0.01) from each other. While F3, F4 (NOV, DEC) showed non-
significant variations from each other but showed highly significant variations from other
fortnights. In AM1 (Amylase 3mg/kg) F3,F4 F5& F6 respectively showed highly significant
(P<0.01) variations from each other also from F1and F2.WhereasF1 and F2 showed non -
significant variations from each other but highly significant from all other fortnights. In AM2
(amylase 6mg/kg) F1 &F2 showed highly significant (P<0.01) variations from other fortnights.
While F3& F4 showed non -significant variations from each other but highly significant from all
other fortnights. F5 and F6 showed highly significant variations from other fortnights but are
non- significant from each other (Table 4.7).

Line chart representation showed maximum value of condition factor was observed in
AM1 (Amylase 3mg/kg) at 2nd fortnight (NOV).While minimum value was observed in control
group at 6th fortnight (Figure 4.5).

Length weight relationship:

It is an important tool to determine the growth of a fish in its environment in response to the
varying levels of enzymes in the diet. After every fortnight increase in average length and weight
were observed. Length is a self-dependent variable while weight depends on it. This relationship
based on the cube law.

W= aL3

W= weight of fish

L= length of fish

a = constant (estimated by regression studies)

A more expensive form of relationship is:

Log W= Log c + n Log L (L = Le Creon, 1951)

c = constant

n = exponent

W =weight (g)

L = length (cm)

Maximum value of r 0.997 showed maximum degree of positive correlation between the length
and weight of Labeo rohita. The value of coefficient of determination near about 1 showed
accuracy of regression model. Minimum value 0.968 was recorded by AM1 (Table 4.8)

r = Correlation coefficient

R² = Coefficient of determination

S.E. Standard Error

X = Total length (independent variable)

Y = Total body weight (dependent variable)

Graphical representation showed that there was an ideal relationship throughout the trial.

Specific Growth Rate

Specific Growth Rate (SGR) is used to address fish growth. This is done using the following
equation: SGR = (ln (final weight in grams) - ln (initial weight in grams) x100) / t (in days)
After the trial of 90 days, highest value of SGR was observed in treatment group AM2 (Amylase
6mg/kg) lagging behind the SGR value 0.81 in AM1 (Amylase 3mg/kg). And the lowest value of
SGR was observed in control group (Table 4.9).

When data was analyzed statistically analysis of variance of treatments showed significant (P<
0.05) variations between the treatments (Table 4.10).Whereas, comparison of means of control
group showed highly significant variations from AM2 and AM1. While AM1 showed non -
significant variation from other treatment groups (Table 4.11).

Bargraph showed the maximum value of SGR in AM2 (amylase 6mg/kg) while minimum
value was showed by control group (amylase 0mg/kg) (Figure 4.7).

Protein efficiency ratio (PER):

At the end of 90 days trial, calculations tells that higher protien efficiency was observed
in AM1 (amylase 3mg/kg) leading the PER value 0.078 of control group. Whereas the lowest
PER was 0.073 in AM2 (amylase 6mg/kg) (Table 4.12)

When data was analyzed statistically analysis of variance showed that treatments have significant
variations among them (Table 4.13) .Comparison of means of AM2 (Amylase 6mg/kg) showed
highly significant variations from control group and AM1 treatment, while AM1 and control
group showed non-significant variations (Table 4.14).

Graphical representation showed the highest value of PER was in AM1 (amylase
3mg/kg) and minimum value was observed in treatment AM2 (Amylase 6mg/kg) (Figure 4.8)

4.2 Nutrient digestibilityCoeffients of Feed

The different analytical analysis used to estimate the effect of different levels of enzymes on fish
digestibility by its composition for different parameters (ash, crude protien, dry matter, gross
energy and lipids) in fish feed.
Moisture:

Highest value of moisture 22% was calculated in feed AM2 (Amylase 6mg/kg)
whichfollowed by feed AM1 (Amylase3mg/kg) with the value 21%, after this control group has
the lowest value of moisture value i.e. 15% (Table 4.15).When data was analyzed statistically
analysis of variance for moisture showed significant (P<0.05) variations among treatments (Table
4.16).While comparison of means for moisture in control group showed highly significant
variations as compared to AM1 and AM2. However AM1(Amylase3mg/kg) and AM2 (Amylase
6mg/kg) showed non- significant (P>0.05) variations from each other (Table 4.17).

Bar graphical representation showed that AM2 (Amylase 6mg/kg) have the highest value
of moisture while control group shows the minimum value (Figure 4.9).

Ash:

Maximum value of Ash content was 79% calculated in AM1 (Amylase 3mg/kg) lagging
behind control group with the percentage of 75%. Minimum value of ash was observed in AM2
(Amylase 6mg/kg) i.e. 74%. When data was analyzed statistically analysis of variance for ash
showed that all the treatments have significant (P<0.05) variations among them (Table 4.18)
When means were compared they showed that there is non-significant variations between control
group and AM2 (Amylase 6mg/kg).Whereas AM1 shows highly significant variations from other
treatments (4.19).

Graphical representation showed the maximum value of ash in AM1 (Amylase 3mg/kg)
and minimum value in AM2 (Amylase 6mg/kg) (Figure 4.10).

Dry matter:

In case of dry matter, control feed have highest value (85%) lifting behind AM1
(Amylase3mg/kg) having the value 79%. Lowest value of dry matter was 78% observed in AM2
(Amylase 6mg/kg). When data was analyzed statistically analysis of variance showed significant
(P<0.05) variations between the treatments (Table 4.20).While comparison of means showed non-
significant variations among AM1 (Amylase 3mg/kg) and AM2 (Amylase 6mg/kg) while control
group showed highly significant variations from other two groups (Table 4.21).

Bar graph showed maximum value of dry matter in control group and minimum value in AM2
(6mg/kg) (Figure 4.11).

Crude protein:

The maximum value of crude protein was showed by AM1 (Amylase 3mg/kg) i.e.
35.93% followed by AM2 (Amylase 6mg/kg) with the value of 34.37%. And the lowest value of
crude protein was 28.12% showed by control group. When data was analyzed statistically
analysis of variance for crude protein showed significant (P<0.05) variations among the
treatments (Table 4.22) .Comparison of means of crude protein control group showed highly
significant variations from AM1 (Amylase 3mg/kg) and AM2 (Amylase 6mg/kg). AM1 & AM2
showed non-significant variations from each other (Table 4.23).

Graphical representation of crude protien showed that AM1 (Amylase 3mg/kg) have
maximum value while minimum value was showed by control group (Figure 4.12).

Crude fat:

Fat content in AM1 (Amylase 3mg/kg) showed the maximum value lagging by AM2 (Amylase
6mg/kg). Minimum value of fat was observed in control group. When data was analyzed
statistically analysis of variance for crude fat showed highly significant (P<0.01) variations
among the treatments (Table 4.24). Comparison of means of crude fat showed that control group
have highly significant variations from other treatments, while there were non-significant
variations among AM1 (Amylase 3mg/kg)and AM2(Amylase 6mg/kg) (Table 4.25).

Bar graph showed minimum value in control group while highest value in AM2 (Amylase
6mg/kg) (Figure 4.13).
Nitrogen free extracts (%):

Nitrogen free extracts in control group showed the maximum value (39.88%) lagging by AM1
(Amylase 3mg/kg) having NFE 25.07%. Minimum value of NEF was 24.63% in AM2 (Amylase
6mg/kg).When data was analyzed statistically analysis of variance showed highly significant
(P<0.01) variations among the treatments (Table 4.26).Whereas comparison of means for
nitrogen free extracts showed that control group have highly significant variations from other
treatments, whileAM1 (Amylase 3mg/kg) andAM2 (Amylase 6mg/kg) showed non -significant
variation among them (Table 4.27).

Graphical representation showed that control group has the maximum value of nitrogen
free extracts while AM1 and AM2 have same minimum value of NFE (Figure 4.14).

Gross energy (Kcal/g-1):

The experimental group control group have the maximum value 482.97 Kcal/g-1 followed
by AM1 (Amylase 3mg/kg) having the value 475.59 Kcal/g -1. Lowest value of gross energy was
474.42 in AM2 (Amylase 6mg/kg).When data was analyzed statistically analysis of variance
showed significant (P<0.05) variations among the treatments (Table 4.28) .While comparison of
means between the treatments showed non-significant variations among control group and AM1
(Amylase 3mg/kg) while AM2 (Amylase 6mg/kg) showed highly significant variations from
other two groups (Table 4.29).

Graphical representation showed that maximum value of gross energy was showed in control
group while the minimum value was shown by AM2 (Amylase 6mg/kg).

Chromic oxide analysis:

Chromic oxide in AM2 (Amylase 6mg/kg) showed the maximum value (0.98%) lagging
by AM1 (Amylase 3mg/kg) and control group having the lowest value 0.96. When data was
analyzed statistically analysis of variance showed significant ( P<0.05) variations between the
treatments (Table 4.30).When means were compared for chromic oxide ,control group showed
non-significant variations from AM1whereas showed highly significant variations from
AM2.However, treatment AM2 showed non- significant variations from AM1 but highly
significant from control group (Table 4.31).
Bar graph showed the maximum value of chromic oxide in AM2 and minimum value in control
group (Figure 4.16).

4.3 Analyzed % of nutrients in fecal matter:

Moisture:

The most noteworthy value of moisture in fecal matter was 6% seen in treatment AM2 (Amylase
6mg/kg), which was followed by the control group with a value of 4.5%. The most minimal
value of dampness in fecal matter was 3.5% observed in AM1 (Amylase 3mg/kg). (Table 4.32)
When data was analyzed statistically, analysis of variance for moisture showed highly significant
(P < 0.05) variations among the treatments (Table 4.33). While correlation of means showed
highly significant (P<0.01) variations when contrasted with AM1 and control group. Whereas
AM1 (Amylase 3mg/kg) and control group showed non-significant (P>0.05) variations from one
another (Table 4.34).

Graphical representation showed highest value in AM2 (Amylase 6mg/kg) and lowest
value in AM1 (Amylase 3mg/kg) (Figure 4.17).

Dry matter:

Maximum value of dry matter was 96.5% seen in AM1 (Amylase 3mg/kg) while the
lowest value was 94 % shown in AM2 (Amylase 6mg/kg) .control group have 95.5% dry matter.
When data was analyzed statistically investigation of fluctuation showed that there were
significant (P<0.05) variations among all treatments (Table 4.35). While correlation of means
AM1 and AM2 showed highly significant variations among themselves but showed non-
significant variations from control group (Table 4.36).

Graphical representation showedlowest value of dry matter in AM2 (Amylase 6mg/kg)

and highest value in AM1 (Amylase 3mg/kg) (Figure 4.18).

Ash:

In case of ash content, AM2 shows the maximum value (76.5) lagging by AM1 (Amylase
3mg/kg) having 76% ash content. Lowest value was 75.5% seen in control group. When data
was analyzed statistically analysis of variance showed critical (P<0.05) variations among
medicines (Table 4.37). While examination of the means AM2 (amylase 6mg/kg) showed highly
significant variations from control group and AM1.Control group and AM1 (amylase 3mg/kg)
showed non- significant variations from each other (Table 4.38).

Barograph representation showed minimum value in control group and highest value in
AM2 (Amylase 6mg/kg) (Figure 4.19).

Crude protein:

The maximum value of crude protein was found in AM2 (Amylase 6mg/kg) 28.12%
followed by AM1 (Amylase3mg/kg) with the value 27.34% while the lowest value of crude
protein was 19.53% found in control group. When data was analyzed statistically analysis of
variance showed significant (P<0.05) variations among the treatments (Table 4.39) .Whereas
examination of means showed highly significant variations in control group from others. While
AM1 and AM2 showed non- significant variations among themselves (Table 4.40).

Graphical representations showed lowest value of crude protein in control group while
highest value of crude protein in AM2 (Amylase 6mg/kg) (Figure 4.20).

Crude fat:

Highest value of fat was 20%found in AM2 (amylase 6mg/kg) and the lowest value
18.78% was showed by control group. While AM1 (Amylase 3mg/kg) have 19.25% crude fat in
it. When data was analyzed measurable investigation of change showed significant (P<0.05)
variations between the treatments for crude fat (Table 4.41).When means were compared control
group and AM2 showed non-significant variations from AM1 while highly significant from each
other ( Table 4.42).

Bar graph representation for crude fat showed maximum value in AM2 (Amylase 6mg/kg) while
lowest value in control group (Figure 4.21).

Nitrogen free extracts:

Nitrogen free extracts in control group shows the maximum value (57.19%) lagging by AM1
(Amylase 3mg/kg) having NFE 49.94%. Minimum value of fat was 45.88 % in AM2 (Amylase
6mg/kg). When data was analyzed statistically examination of fluctuation for nitrogen free
concentrates showed significant (P < 0.05) variations among treatments (Table 4.43). While the
correlation of means between AM2 and control group shows highly significant variations from
each other but are non -significant from AM1( Table 4.44).

Graphical representation showed maximum value in control group while least value in AM2
(Amylase 6mg/kg) (Figure 4.22).

Gross energy (Kcal/g-1):

Peak value 541.16 Kcal/g-1 of gross energy was found in AM1 (Amylase 3mg/kg) lagging
by AM2 (Amylase 6mg/kg) with the value 535.98 Kcal/g-1. Lowest value of gross energy was
522.47 Kcal/g-1 found in control group. When data was analyzed statistically analysis of
variance showed significant (P<0.05) variations among the treatments (Table 4.45) While
comparison of means showed non-significant variations between AM1 (Amylase 3mg/kg)
andAM2 (Amylase 6mg/kg) while highly significant variations were observed between control
and treatment groups (Table 4.46).

Bar graph representation showed least value in control group while maximum value in
AM1 (Amylase 3mg/kg) (Figure 4.23).

Chromic oxide analysis:

Chromic oxide analysis in AM1 and AM2 showed the maximum value (1.41 %) lagging
by control group having chromic oxide 1.37 %. When data was analyzed statistically analysis of
variance for chromic oxide showed significant (P<0.05) variations between the treatments (Table
4.47) whereas comparison of means showed non-significant variations between AM1 (Amylase
3mg/kg) andAM2 (Amylase 6mg/kg) while highly significant variations were shown between
control and treatment groups (Table 4.48).

Graphical representation showed maximum value of chromic oxide in AM1 (Amylase

3mg/kg) while lowest in control group (Figure 4.24).

4.4 Approximate nutrient utilization

Moisture:

The most extreme value of moisturein approximate nutrient utilization in Labeo rohitawas 5%,
seen in AM2 (Amylase 6mg/kg) and in the control group. While the base value was 4.9% as seen
in AM1 (Amylase 3mg/kg).When data was analyzed statistically, analysis of variance showed
non-significant (P>0.05) variations among the treatments (Table 4.50). When means were
compared, there were non-significant variationsamong the treatments (Table 4.51).

Bar graph representation showed maximum value for moisture in AM2 (Amylase 6mg/kg) and in
control group while least value was showed by AM1 (Amylase 3mg/kg)(Figure 4.25).

Dry matter:

Maximum value of dry matter was 96.2% seen in AM1 (Amylase 3mg/kg) while the
lowest value was 93% showed by AM2 (Amylase 6mg/kg).Control group have 95% moisture.
When data was analyzed statistically analysis of variance showed significant (P<0.05) variations
between the treatments (Table 4.52) .When means were compared there was non-significant
variations between AM1 (Amylase 3mg/kg) and control group while AM2 (Amylase 6mg/kg)
showed highly significant variations from other treatments (Table 4.53).

Graphical representation AM1 showed maximum value of dry matter and AM2 showed
minimum value of dry matter (Figure 4.26).

Ash:

In approximate nutrient utilization, highest ash content 85.2% was observed in control group
while lowest value was 82% observed in AM2(Amylase 6mg/kg).Ash content in AM1(Amylase
3mg/kg) was 84%. When data was analyzed statistically examination of change showed highly
significant (P<0.01) variations among treatments (Table 4.54). While comparison of means
control group, AM1 and AM2 showed highly significant variations among themselves (Table
4.55).

Bar graph representation of ash showed the maximum value in control group and minimum value
in AM2 (Amylase 6mg/kg) (Figure 4.27).
Crude protein:

The maximum value of crude protein was found in AM2 (Amylase 6mg/kg) i.e. 39.4%
followed by AM1 (Amylase 3mg/kg) with the value 32.4% while the lowest value of crude
protein was 30.54% found in control group. When data was analyzed statistically analysis of
variance showed highly significant (P<0.01) variations between the treatments (Table 4.56).
However,comparison of mean showed non -significant P>0.05) variations between control group
and AM1 (Amylase 3mg/kg) while AM2 showed highly significant (P<0.01) variations from
AM1and control group (Table 4.57).

Graphical representation showed highest value of crude protein in AM2 and lowest value in
control group (Figure 4.28).

Crude fat:

Highest value of fat was 15%found in AM1 (Amylase 3mg/kg) and the lowest value
10.57% was showed by control group and AM2 (Amylase 6mg/kg) have 11.4% crude fat in it.
When data was analyzed statistically analysis of variance showed significant (P<0.05) variations
between the treatments (Table 4.58).When means were compared they showed highly significant
variations in AM1 as compared to control group and AM2.Whereas AM2 (Amylase 6mg/kg) and
control group showed non- significant variations from each other (Table 4.59).

Bar graph representation for crude fat showed maximum value in AM1 (Amylase 3mg/kg) while
minimum value in control group (Figure 4.29).

Nitrogen free extracts:

Highest value of NFE was 27.8%found in AM1 (Amylase 3mg/kg) and the lowest value
25.07% was showed by control group.AM2 (Amylase 6mg/kg) have 27% NFE in it. When data
was analyzed statistically analysis of variance showed highly significant (P<0.01) variations
among the treatments for NFE (Table 4.60).While comparison of means for NFE showed non-
significant variations between AM1 and AM2.Control group showed highly significant
variations from other treatments (Table 4.61).

Graphical representation for NFE showed least value in control group while AM1 (Amylase
3mg/kg) have highest value of NFE (Figure 4.30).
Gross energy (Kcal/g-1):

Peak value 626.5Kcal/g-1 of gross energy was found in AM1 (Amylase 3mg/kg) lagging
by AM2 (Amylase 6mg/kg) with the value 606.5Kcal/g-1. Lowest value of gross energy was
575.3Kcal/g-1 found in control group. When data was analyzed statistically analysis of variance
showed significant (P<0.05) variations within the treatments (Table 4.62).Whereas comparison
of means control group and AM1 showed highly significant variations from each other but
showed non-significant variations from AM2 (Table 4.63).

Graphical representation showed least value of gross energy in control group while
highest value in AM1 (Amylase 3mg/kg) (Figure 4.31).

Apparent digestibility coefficient (%):

Dry matter:

In apparent digestibility coefficient the greatest amount of dry matter was 21.6% found in the
control group while slacking by 18% in AM1 (Amylase 3mg/kg) and AM2 (Amylase 6mg/kg).
When data was analyzed statistically analysis of variance showed highly significant (P <0.01)
varieties among the treatments (Table 4.65). When comparison of mean was done AM1 and
AM2 showed non-significant variations between them. The control group showed highly
significant variations from other groups (Table 4.66).

Graphical representation showed that maximum value of dry matter was in control group
while AM1 and AM2 have same lowest value (Figure 4.32).

Ash:

In apparent digestibility coefficient, highest ash content 35% was observed in AM1 (3mg/kg)
while the lowest value 29% was observed in AM2 (6mg/kg) and control group have 30% ash
content. When data was analyzed statistically analysis of variance showed significant (P<0.05)
variations between the treatments (Table 4.67).While comparison of means showed non-
significant variations among control group and AM2while AM1 showed highly significant
variations from other treatments (Table 4.68).

Graphical representation for ash showed maximum value of ash inAM1 (Amylase
3mg/kg) and lowest value in AM2 (Amylase 6mg/kg) (Figure 4.33).

Crude protein:

The maximum value of crude protein was found in AM1 (Amylase 3mg/kg) i.e. 49%
followed by AM2 (Amylase 6mg/kg) with the value 45% while the lowest value of crude protein
was 0.1% found in control group. When data was analyzed statistically analysis of variance
showed significant (P<0.05) variations among the treatments (Table 4.69).When means were
compared control group and AM1 are highly significant from each other but are non-significant
from AM2 (Table 4.70).

Graphical representation showed least value for crude protein in control group and
highest value for crude protein in AM1 (Amylase 3mg/kg) (Figure 4.34).

Crude fat:

Highest value of fat was 46% found in AM1 (Amylase 3mg/kg) and the lowest value 23% was
showed by control group. And AM2 (Amylase 6mg/kg) have 41% crude fat in it. When data was
analyzed statistically analysis of variance showed highly significant (P<0.01) variations among
the treatments (Table 4.71). When means were compared control group shows highly significant
variations from other treatments AM1 and AM2 while AM1 and AM2 showed non-significant
variations from each other (Table 4.72).

Barograph representation showed maximum value of crude fat in AM1 and least value in
control group (Figure 4.35).

Gross energy (Kcal/g-1):

Peak value 24.4Kcal/g-1 of gross energy was found in control group lagging by AM1
(Amylase 3mg/kg) with the value 24Kcal/g-1. Lowest value of gross energy was 11Kcal/g -1 found
in AM2 (Amylase 6mg/kg). When data was analyzed statistically analysis of variance showed
highly significant variations (P<0.01) among the treatments (Table 4.73). Whereas, comparison
of means AM2 (Amylase 6mg/kg) shows highly significant variations from control group and
AM1.While, AM1 and control group have non-significant variations from each other (Table
4.74).

Graphical representation control group and AM1 showed almost same maximum value of
gross energy while AM2 showed lowest value (Figure 4.36)

4.6 Analyzed percentage of nutrients in body meat:

Moisture:

Peak value of moisture 5.5% was determined in body meat AM1 (Amylase 3mg/kg), lagging
behind the control group with a value of 4.5%. After this, AM2 (Amylase 6mg/kg) has the least
amount of dampness, 3.5%. When data was analyzed statistically, analysis of variance showed
significant (P < 0.05) variations among the treatments (Table 4.76). While comparison of mean
AM1 and AM2 showed highly significant variations from one another yet were non-significant
from the control group (Table 4.77). TABLES? FIG?

Graphical representation formoisture showed lowest valuein AM2 and highest value in
AM1 (Amylase 3mg/kg) (Figure 4.37).

Dry matter:

In case of dry matter, body meat AM2 (Amylase 6mg/kg) comes up with the highest value
(96.5%) lagging by control group having the value 95.5%. Lowest value of dry matter was
94.5% observed in AM1 (Amylase 3mg/kg). When data was analyzed statistically analysis of
variance showed significant (P<0.05) variations between the treatments (Table 4.78). Whereas
comparison of means showed non-significant variations in control group from AM1 and AM2
while AM1 and AM2 showed highly significant variations from each other (Table 4.79).

Graphical representation showed low value in AM1 (Amylase 3mg/kg) and high value in
AM2 (Amylase 6mg/kg) (Figure 4.38).

Ash:
 Maximum value of Ash content was 84.5% calculated in control group lagging by AM2
(Amylase 6mg/kg) with the percentage of 83.5%. Minimum value of ash was observed in AM1
(Amylase 3mg/kg) i.e. 83%. When data was analyzed statistically analysis of variance showed
significant (P < 0.05) variations between the treatments (Table 4.80).While comparison of
means, control group and AM1 showed highly significant variations from one another, while
showing non-significant variations from AM2. (Table 4.81)

Graphical representation for ash percentage showed minimum value in AM1and

maximum value in control group (Figure 4.39).

Crude protein:

The maximum value of crude protein was showed by AM2 (Amylase 6mg/kg)i.e.33.55%
followed by AM1 (Amylase 3mg/kg) with the value of 32.03%. The lowest value of crude
protein was 24.21% showed by control group. When data was analyzed statistically, analysis of
variance showed significant (P<0.05) variations between the treatments (Table 4.84).When
means were compared AM1 and AM2 showed non-significant variations from each other while
control group showed highly significant variations from other groups (Table 4.83).

Barograph for crude protein showed minimum value in control group and highest value
in AM2 (Amylase 6mg/kg) (Figure 4.40).

Crude fat:

Fat content in control group and AM1 (Amylase 3mg/kg) shows same maximum value
(7.75%). Minimum value of fat was 6.7% inAM2 (Amylase 6mg/kg). When datawas analyzed
statistically, analysis of variance showed significant (P<0.05) variations between the
treatments(Table 4.84).While, comparison of means showed non-significant variations between
control group and AM1 while AM2 showed highly significant from other treatments (Table
4.85).

Graphical representation showed lowest value in AM2and highest value in control group (Figure
4.41).
Nitrogen free extracts (%):

The maximum value of NFE was showed by control group i.e.63.53% followed by AM2
(Amylase 6mg/kg) with the value of 56.21%. The lowest value of NFE was 54.72% showed by
AM1 (Amylase 3mg/kg). When data was analyzed statistically analysis of variance shows highly
significant (P<0.01) variations between the treatments (Table 4.86) When means were compared
for NFE control group showed highly significant variations from AM1 and AM2 while AM1 and
AM2 showed non-significant variations from each other ( Table 4.87).

Graph showed maximum value of NFE in control group and lowest value of NFE in
AM1 (Figure 4.42).

Gross energy (Kcal/g-1):

The experimental group AM2 (Amylase 6mg/kg) have the maximum value 483.7Kcal/g-1
followed by AM1 (Amylase 3mg/kg) having the value 478.7Kcal/g-1. Lowest value of gross
energy was 470.85 in control group. When data was analyzed statistically,investigation of
difference showed significant (P<0.05) variations among the treatments (Table 4.88).While,
correlation of means control group and AM2 showed highly significant variations from one
another while non-significant from AM1 (Table 4.89).

Bar graph showed minimum value of gross energy in control group and highest value in AM2
(Amylase 6mg/kg) (Figure 4.43)

4.7 Digestive Enzyme Assay (U mg-1):

Digestive enzyme assay (U mg-1) of Labeo rohitareared under control and different
treatment groups.

Digestive enzyme assay in liver for amylase showed greatest value in AM2 (3.51U mg-1)
slacking by AM1 (2.83 U mg-1).While control group has least value of 0.91 U mg-1. However in
the gut digestive enzyme activity for amylase showed the greatest worth in AM2 (2.89 U mg-1)
slacking by AM1 with a value of 2.58 Umg-1.Whereas,control group has the least value of(0.77
U mg-1) (Table 4.90).
When data was analyzed statistically, analysis of variance showed highly significant variations
among all the treatments (Table 4.91). When comparison of mean was done there werealso
highly significant variations between all the treatments (Table 4.92).

Bar graph showed highest value was observed in AM2 (Amylase 6mg/kg) and least value was
observed in control group (Figure 4.44).

When data was analyzed statistically analysis of variance for gut indicates that the control and all
the treatments were highly significant (P<0.01) from each other (Table 4.93).When means were
compared they showed non-significant variations between AM1 (Amylase 3mg/kg) and AM2
(Amylase 6mg/kg) while control group showed highly significant from other two groups (Table
4.94).

Bar graph representation showed maximum value in AM2 (Amylase 6mg/kg) and lowest
value in control group (Figure 4.45).

DISCUSSION

Exogenous enzyme was added to the fish meal, which showed favorable results. In this study,
varying levels of enzyme amylase are added to the diet to see the possible effect on Labeo rohita
fish's growth performance, body composition, digestive enzyme activity and nutrient
digestibility. A 90-days trial was conducted. During the trial, the mortality rate was nil,
indicating that the rearing circumstances were appropriate.

Growth parameters

The average weight and length of Labeo rohita were measured every two weeks during the
current growth research. The experimental groups perform better than the control group when
enzymes were supplemented. The highest growth was shown in the experimental groups AM1 (3
mg/kg) and AM2 (6 mg/kg). Increases in growth parameters caused by enzymes in fish feed
imply a link between weight gain and enzyme consumption. It can be mentioned that using
enzymes is necessary for acquiring the maximum weight. Enzymes serve a crucial function in
speeding up chemical reactions, allowing the fish to perform better.

Exogenous enzyme in the meal lowered anti-nutritional factors while also assisting endogenous
enzyme in performing better and increasing digestion (Gomes et al., 2016).

According to present study supplementation of enzyme amylase in fish feed showed pronounced
effect on growth performance of Labeo rohita. Similar results were also found by Ghafarifarsani
et al. (2022) in Cyprinus carpio according to his findings, fish supplemented with diet
containing amylase shows greater final weight ,weight gain(WG) and specific growth
rate(SGR).Similar observations were also made by (Sangavi, Sawant et al. 2020) who reported
that amylase increase the growth performance in Labeo rohita at optimal level.

Similar results were also seen by (Sajina et al., 2019) in Labeo rohita, according to his study,
amylase has a growth-promoting effect without degrading the immune-modulating
function .Study conducted by Patil et al.(2019) the exogenous enzyme amylase caused an
increase in weight gain, maximum length, and specific growth rate.

Yigit et al. (2018) reported some conflicting results that the exogenous enzymes did not increase
the growth when added to the fish feed. In the report of many researchers supporting results
were found who reported the positive effects of enzymes complex on the growth of
catlacatla,Labeorohita and Cyprinus carpio. Another researcher Khalil et al. (2018), according
to his report all groups supplied with enzyme (amylase) had greater condition factors, specific
growth rates, and FCR than the control group.

Due to amylase activity increase in weight and length was also found by (Umalatha, et al. 2016)
due to activity of amylase.Diffrent findings were also reported by Maiti et al. (2019) who found
that exogenous enzyme supplementation in leaf-based fish diet did not significantly improve the
growth performance of fish when compared to non-supplemented feed.

Study conducted by Olude et al. (2021) reported the maximum increase in growth performance
due to amylase present in the diet of L.rohita. Sadek et al. (2021) reported that enzyme
supplementation (Amylase, protease) improve growth performance, health and feed utilization of
fish. All these studies proved that enzyme supplementation in fish feed had striking results.

From the same enzyme different researchers found the dissimilar outcomes by cause of different
factors like different fish species, experimental designs, environmental conditions and different
dose level of enzyme

Apparent nutrients digestibility coefficient (%)

This experimental study showed that the enzyme amylase promote the digestibility and
utilization of diet. Sadek et al. (2021) reported that enzyme supplementation (Amylase, protease)
improve growth performance, health and feed utilization of fish.

Similar results were found by Guan et.al. (2021) that apparent digestibility of dry matter was
significantly improved due to amylase activity. Another scientist Nottanalan et.al (2021)performs
experiment on Labeo rohita and reported that amylase enzyme in feed increase the dry matter
digestibility coefficient. Opposite results were shown by Kumar et.al (2018) that enzyme
supplementation in fish diet does not affect the nutrient utilization.

Apparent digestibility coefficient of protien was higher in AM1 (amylase 3mg/kg) comparable
results were reported by Das et al. (2021) amylase activity increases the crude protien utilization.

Another scientist Sangavi et al. (2020) revealed the same results in juvenile Labeo rohita that
activity of amylase in feed enhance the protien utilization. Contrasting results were observed by
Gai et al. (2022) that enzyme administration does not enhance the protien utilization.

The ADC of fat was maximum in AM1 (amylase 3mg/kg) similar trend was observed by Javid et
al. (2021) who conducted experiment on Labeo rohita which shows maximum increase in crude
fat utilization due to supplementation of enzyme amylase. comparable results were reported by
Das et al. (2021) amylase activity increases the crude fat utilization.

ADC of gross energy was higher in AM1 (amylase 3mg/kg) same observation was done by Lee
et al. (2020) who found that the supplementation with the amylase enzyme increase the gross
energy value of a fish.

….
Body composition

This experimental study showed that percentage of body composition was observed maximum in
the amylase group. Similar results were found by Abbas et al. (2021) who perform experiment
on Labeo rohita and found better body composition due to amylase activity.

Another scientist Jimoh et al. (2019) conduct experiment on lemon fin barb in which amylase
showed superior body composition as compared to control diet. Opposite results were found by
Gopan et al. (2020) who perform a trial on Labeo rohita and showed that activity of digestive
enzyme amylase did not affect the body composition of fish.

Similar results were revealed byPhulia et al. (2018) that fish had higher body composition due to
amylase in diet. Sadek et al. (2021) also observed the same finding that exogenous enzyme
enhance the body composition of fish. Same trend was observed by Imani et al. (2017) who
found the higher ash content because of amylase activity in rainbow trout.

Different researcher observed different results from the same enzyme which is due to different
factors like environmental conditions, experiment design, use of different fish and use of
different enzyme level.

Digestive enzyme activity

Present research study revealed that digestive enzyme activity of gut and liver for amylase
showed maximum value. Maximum value was found in AM2 (amylase 6mg/kg).

Similar results were indicated by Kong et al. (2021) who observed improvement due to digestive
activity of amylase in liver and gut of Channaargus. Another researcher Mass et al. (2021)
showed that exogenous enzyme supplementation enhances the digestibility in middle and
proximal gut. Murtaza et al. (2016) observed the activity of digestive enzymes in
Hypophthalmichthys molitrix, CatlacatlaandCyprinuscarpio fed corn gluten. The liver had
higher amylase activity than the intestine.
Contradictory results were found by Yengkokpam et al. (2022) who observed that amylase did
not affect the digestive enzyme activity of gut and liver of rohu.

Chapter 5

SUMMARY

Present experimental study entitled as Evaluation of enzyme additives (amylase) on the

growth, apparent nutrients digestibility, body composition and digestive enzyme assay of Labeo
rohita” was conducted at the fisheries research lab, Department of zoology, Government College
University Faisalabad.

For the accomplishment of this feeding trial, 200 fingerlings were acquired from Punjab
Fish Hatchery Santayana road Faisalabad. Before starting this experiment, fingerlings were
acclimatized for one week to the laboratory condition by feeding them on the control diet (32%
crude protein) once in a day at 4% live wet body weight. After acclimatization, fingerlings were
divided into six fish tanks at 20 fingerlings in each tank with one replicate. One control and two
experimental diets were prepared viz. T1: amylase: 3mg/kg, T2: amylase: 6mg/kg. Water
exchange was done manually at daily basis and oxygen was supplied trough the oxygen pumps.

Weight and length was calculated at fortnight basis as fish feeds on experimental diet.
For the measurement, 10 fingerlings were randomly selected from each tanks and allowed to
electronic weight balance for the measurement of weight and for the length centimeter scale was
used. After two hours of feeding session fecal matter was collected and dried in oven at 60 oC
and put away in impenetrable containers. Toward the finish of the trial, fish fingerlings were
broiler dried, crushed, and packed in the jars. Different procedures were performed for the
estimation of ash, crude fat, dry matter, gross energy, crude protein and chromic oxide.Liver and
gut was extracted from the three fingerlings randomly of each treatment for the digestive enzyme
assay. Supernatants were made and stored at -20C; and amylase activity was measured by the
starch hydrolysis method.

Labeo rohita fingerlings showed maximum growth in the AM1 amylase 3mg/kg as
compare to other groups. Maximum and minimum increase in weight was observed as 4.8g and
0.1g during 2nd and 5th fortnight. The specific growth rate showed maximum value in AM2
amylase 6mg/kg.

The apparent digestibility coefficient of dry matter and protein was observed maximum
in AM1 amylase 3mg/kg whereas the ADC of ash, lipid and gross energy was observed
maximum in Am1 amylase 3mg/kg.

The percentage of nutrients in body composition for dry matter, crude protien, nitrogen
free extract and gross energy was higher in AM2(amylase: 6mg/kg) while nutrients in body
composition for moister and crude fat was maximum in AM1(amylase: 3mg/kg).However
maximum value of ash was observed in control group.

The approximate nutrients utilization values for dry matter, crude fat , chromic oxide and
gross energy were higher in the Am1 (amylase 3mg/kg) while crude protein showed maximum
value in AM2(6mg/kg). Maximum value of Ash was observed in the control group.

The digestive enzyme activity of liver and gut for amylase was found maximum in AM2
(amylase: 6mg/kg).

Conclusions and Recommendation:

 The present study proved that amylase enzyme supplementation play a remarkable role in
fish feed enhances the growth of Labeo rohita. Also, its different levels have striking effect on
the digestibility enzyme assay and nutrients digestibilityLabeo rohita fish. To give awareness
about enzyme implementation in fish feed a specific area was design for fish farmers so they can
improve their fish production and quality.

REFFERENCES??????????????