faster af pi{tl000pl{^ - COnnecting REpositories · 2018. 1. 4. · supervision by Ms. Syeda Sadaf. The work is original and has been pursued by the candidate independently. It embodies

EVALUATION OF LEVELS AND SOURCES OF LIPIDS FOR INCLUSION IN THE DIETS OF

FINGERLING CIRRHINUS MRIGALA

DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR

THE AWARD OF THE DEGREE OF

faster af pi{tl000pl{^ IN

- ZOOLOGY

By

SYEDA SADAF

Under the Supervision of

DR. MUKHTAR A. KHAN

FISH NUTRITIOON RESEARCH LABORATORY DEPARTMENT OF ZOOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2008 ^

' * * • " ' 1 ^ fi-

r External : 2700920/21-3430 P " ° " « » \ Internal : 3430

D E P A R T M E N T O F Z O O L O G Y ALIGARH MUSLIM UNIVERSITY

„ ^ ALIGARH-202002 1. ENTOMOLOOY INDIA 2. FISHCRY SCIENCE « AQUACUt.TURE 3. QENETIC* 4. NEMATOUXIY 5. PARASITOLOQY

D. No. JZD

Datad..

Certificate

This is to certify that the dissertation entitled "Evaluation of levels and sources of lipids for

inclusion in the diets of fingerling Cirrhinus mrigala" has been completed under my

supervision by Ms. Syeda Sadaf. The work is original and has been pursued by the candidate

independently. It embodies some interesting observations contributing to the existing knowledge

on the subject. I permit the candidate to submit the work for the award of degree of Master of

Philosophy in Zoology of the Aligarh Muslim University, Aligarh, India.

t^A.lcl^

X>r. ZM^u^tar < ^ j\Jian

Reader

CONTENTS Page No.

ACKNOWLEDGEMENTS

GENERAL INTRODUCTION 1-10

GENERAL METHODOLOGY 11-18

CHAPTERS

1. Effects of dietary lipid levels on growth, conversion efficiency and body

composition of fingerling mrigal, Cirrhinus mrigala.

19-42 2. Effects of different lipid sources on growth, conversion efficiency and

body composition of fingerling mrigal, Cirrhinus mrigala.

43-52

REFERENCES 53-94

ACKXOM^L'ECDgM'EmS

(First of all I Bow my head in reverence to Almighty AO^h the omnipotent and

omnipresent for 9fis Blessings which provided me enough zeal to complete this wor^

I am highty indsBtedto <Dr. Mu^tarA- "Kfian, my respected supervisor for

the vaJked guidance during my study, giving me plenty of his time, talents,

^jurwkdge and patience, his experience and human qualities who spent time and

effort improving nr^professionals^iOs, giving valuaBle suggestions and discussions,

which have Been decisive for the completion of this (Rssertation. I shall (dways

rememBer his painsU^ng interest, patience and constant encouragement despite all

my lapses and shortcomings.

I wouldpartictUdrly G^ to than^(Prof ABsar Mustafa lOian, Chairman,

(Department of Zoology and (Dean TacuHy of Lfe science for providing necessary

QiBoratoryfacilities.

I would B^ to put on record my heartfelt than^ to ^rof AsifA- %hdn,

(Prof IqBal (Parvez, (Dr. fWdfi IrshaduQah and (Dr. ABBas ABidi, for giving me

encouragement and inspirations during the course of research.

I express my than^ to my seniors 9is. S. Tatma ABidi,, Ms. TaBassum Q.

Siddiqui and Ms. Tarhat.

I feel pleasure to express my special than^ to my Best friend and laB

colleague SeemaB. I ta^ this opportunity to acknowledge the help, constructive

criticism, fieCpfuC cufvice, constant encouragement and inspiration that have Been

instrumentafin overcoming the complexities at every step of this wor^

I am aCso than^fuf to my friends !Hd6ee6a, Matyam and ^uzhat, for

constant encouragement andmoraCsupport throughout the wor^

Last But not least, I e^ess my deepest gratitude to it^JiBBu andjimmifor

their everCasting support, affection forBearance xvithout which it would have Been

impossiBle for me to accomplish the tas^of completing this wor^ Their prayer and

sacrifices help me to gain this siuxesss. I would G^ to express special than^ to my

Brothers andBhaBhifor their constant encouragement.

Lastly my love, regards and Best wishes to aO^ well wishers Qimin)

GENERAL INTRODUCTION

INTRODUCTION

During the last fifty years, enormous growth of human population has taken place all over the

world, especially in developing countries and has crossed thousand million in India. Although

agricultural production has also increased substantially but per capita availability of food is

still low and there is shortage of protein, fat and calorie in diet of a man. Many of the world

poor and low income people still lack access to enough food to sustain their health and normal

daily labors. The absolute numbers of food insecure people are growing annually (Williams,

1997). Hunger and malnutrition remain amongst the most devastating problems facing the

world's poor. Tragically, a considerable portion of the global po})ulation is currently suffering

from one or more forms of nutrient deficiencies (FAO, 2003^).

Because of increasing population the land available for agriculture is shrinking, so there

is need to look for alternative resources of food production, such as the ocean, because they

contain millions of billions of tons of seafood that is plentiful source of high quality protein.

India has an impressive array of aquatic resources of capture and culture potential: approx.

8000 km of coastline, 2.02 million km^ of EEZ, 2 million hectare of potential brackish water

culture areas, 2.05 million hectare of reservoirs, 2.254 million hectare of freshwater ponds and

1.3 million hectare of oxbow lakes and derelicts waters (FAO, 2001^).

Fish can make a imique contribution to improve and diversify dietary intake and promote

nutritional well-being among most population groups. Fish and other forms of sea food can be

used to provide balanced diet and reduce the protein gap. Fish have a highly desirable nutrient

profile providing an excellent source of high quality animal protein containing all the ten

essential amino acids in desirable concentrations that are easily digestible and of high

biological value (FAO, 2003*'). Consumption of fish enhances brain development and learning

in children, protects vision, offers protection from cardiovascular disease and also some

cancers. The fats and fatty acids in fish, particularly the long chain n-3 fatty acids (n-3 PUFA),

are highly beneficial and are difficult to obtain from other food sources. Of particular

importance are eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3,

DHA). Lipids from these marine oils also can have beneficial effects on cardiovascular health.

Fish also contains vitamin A and D and several minerals like calcium, magnesium, sodium,

phosphorus, iron, copper, etc. which constitute 1-2%. Fish, therefore, can provide an important

source of nutrients particularly for those whose diets are monotonous and lacking in animal

products. Fish is not only a vital food, it is also the source of work and money for millions of

people around the globe. It has been estimated that over onf'nillion people rely on fish as their

primary source of protein and some 120 million people are employed in fishery related job

worldwide (FAO, 2006).The world production of convention fish varieties is nearing an almost

saturation level and may even exceed the maximum sustainable yield by 2015. The world

marine fish landings is expected to be leveling somewhere around 90 million tones/year (FAO,

2007^). At the same fime, there has been a continuous increase world wide in demand for both

finfishes and shellfish due to rapidly growing population-'and concern over the high quality

proteinaceous food. This increases the gap between supply and demand for fish, thereby

threatening national food security in many countries.

The only solution for this problem is aquaculture which gives surprising boost to fish

production. It is generally accepted that aquaculture has the greatest potential to bridge this gap

as capture fisheries, though still the major fish source in most countries, are static or in decline

due to over-fishing. It has a significant positive contribudon to food security through extensive

and semi-intensive systems of production. The Global production offish from aquaculture has

grown rapidly during the past four decades, contributing significant quantities to the world's

supply of fish for human consumption. Aquaculture now accounts for nearly half (45 percent)

of the world's food fish and this increase is expected to reach 50 percent in 2015. Aquaculture

has the potential to meet the growing global demand for nutritious food fish and to contribute

to the growth of national economies, while also supporting the sustainable livelihoods of many

communities (FAO, 2007*'). Aquaculture is the fastest growing food-producing industry

(NACA/FAO, 2000.1t can also make significant contributions to development by improving

incomes, providing employment opportunities and increasing the returns on resource use. It

significantiy contributes to the national GDPs in many developing countries in Asia and Latin

America.

Successful and sustainable aquaculture depends upon the provision of nutritionally

balanced, environmental friendly and economically viable practical feeds. Feeds and feeding

are the crucial elements in the culture of aquatic animal. Therefore, knowledge on nutrition and

practical feeding of fish is essential for successful aquaculture. In aquaculture operations, feed

accounts for more than one-half of the variable cost (Akiyama and Chwang, 1989;

Chamberlain, 1996; D'Abramo and Sheen, 1996; Han et al., 2004; Cortes-Jacinto et al., 2005;

Abimorad and Cameiro, 2007; Piedecausa et al., 2007; Martins et al., 2007), often ranging

from 50-60% depending upon the intensity of operation and any reduction in the feed cost

either through their development, improved husbandry or other direct or indirect means is,

therefore, crucial to the development and well being of the industry. Any balanced formula for

fish diet should include an energy source plus a good balance of energy and non-energy, amino

acids, essential fatty acids, specific vitamin and minerals to support life and promote growth

(Halver, 2002).

Protein is the most important nutrient affecting growth performance and feed cost (Kim

et al., 2006). The optimal protein utilization is closely related to its concentration in the diet

and the availability of dietary non-protein energy sources, such as lipids and carbohydrates (El-

Sayed and Garling, 1988; Kaushik and Medale, 1994; Chou and Shiau, 1996; Nankervis et al.,

2000; Watanabe et al., 2001). It is important to provide an adequate level and ratio of protein,

lipid and carbohydrate in diets in order to reduce cataboli:m of protein for energy. Therefore,

the protein and energy concentrations must be balanced for maximizing growth potential (Ellis

and Reigh, 1991). An adequate supply of energy should be provided from non-protein energy

sources such as lipid and carbohydrate. This can minimize the use of protein, thereby, reducing

the overall cost offish production, as protein is the single most expensive ingredient in finfish

diets (Ai et al., 2004).

Amongst the non-protein energy sources in feed, lipid deserves special attention due to

its high calorific value than carbohydrates. Since lipid contains approximately twice as much

gross energy as protein and carbohydrates, they contribute greatly to energy levels, even when

present in relatively low quantities in the diet (Anwar and Jafri, 1995). Dietary lipids besides

providing energy, serve as a source of essential fatty acids (Watanabe, 1982) needed for

structural maintenance of membranes and proper functioning of many physiological processes.

They are well metabolized by fish and are important to achieve good growth rates and

development (Martino et al., 2002). It also provide vehicle for absorption of fat-soluble

vitamins and sterols (NAS-NRC, 1983). Lipids especially phospholipids and sterol esters, play

a vital role in structure of biological membrane both at cellular and sub-cellular level. The long

chain fatty acids (PUFA) are precursor for prostaglandins in fish, which exhibit hormone like

activities. The reduction of protein level in the feed formulation and increase in the dietary

lipid level to compensate the protein for energy requirement not only increases the fish growth

but also reduces the feed cost (Phillips et al.. 1969; Marimuthu and Sukumaran, 2001; Borba et

al., 2006). Feeds with sufficient dietary lipids allow maximal protein sparing (Watanabe et al.,

1979; Garcia et al., 1981; Beamish and Medland, 1982; Sargent et al., 1989; Calduch-Giner et

al.,1999; Ellis and Reigh, 1991; Peres and Oliva-Teles, 1999; Fu et al., 2001; Ai et al., 2004;

Skalli et al., 2004; Kim and Lee, 2005; Ovie et al., 2005; Babalola and Apata, 2006; Fu and

Cao, 2006; Schulz et al., 2007). Protein sparing by dietary lipid implies less discharge of

nitrogen and phosphorus compound into the aquatic environment, providing more economic

and eco-friendly diets (Carter and Hauler, 2000; Hernandez et al., 2004). Lipid imparts flavour

and textural properties to the feed consumed by fish. High lipid concentrafion in feed pellets

contributes to stability of feeds in water (Chaiyapechara et al., 2003). The use of non-protein

energy in fish diets, however, must be closely evaluated as its excessive use can reduce the

feed intake, produce fatty fish and inhibit the utilization of other nutrients (Ali and Al-Asgah,

2001).

In addition to lipid level, dietaiy lipid sources must also be evaluated carefully.

Because besides its amount, qualitative properties also influences the quality of product

(Molnar et al., 2006). Moreover, lipid nutrition of fish and crustaceans, such as lipid sources,

has aroused interest of aquaculturists and nutritionists because of their effect on growth and

health of the organism (Deering et al., 1997; Sargent et al., 2002). Like other vertebrates, fish

are unable to synthesize long chain PUFA de novo and must obtain via the food. It is presumed

that, coldwater fish have a greater nutritional requirement for n-3 fatty acid but warm water

fish, have a greater requirement for n-6 fatty acids found more in vegetable oil than n-3 fatty

acid found in fish oil, for maximal growth (NRC, 1993). The essential fatty acid requirement of

some fireshwater fishes like carp can be satisfied by a mixture of n-6 and n-3 fatty acids

(Castell, 1979; NAS-NRC, 1983).

The main lipid source employed in fish feeds is fish oil, due to its traditionally good

availability and high content in n-3 HUFA (polyunsaturated fatty acids of the n-3 series with

20 or more carbons). It has been estimated that about 60% of the global supply of fish oil is

used in aqua feeds and by the year 2010, fish oil used in aquaculture will be about 75% of the

world supply (Barlow, 2000). Moreover, in the past decade, global fish oil production has

reached a plateau because of over fishing. Therefore, it has become an increasingly urgent

issue to find alternative for fish oil for use in aquaculture feeds. Since, fish oil is expensive

item of the feed, the use of less expensive alternative oil sources such as vegetable or animal

fat for partially or totally replacement offish oil is an important research interest in aquaculture

nutrition. The replacement of fish oil with other lipid sources appears to be possible when the

essential fatty acid (EFA) requirements are satisfied (Hardy et al., 1987; Sargent et al., 1999;

Caballero et al., 2002; Turchini et al., 2003; Richard et al., 2006; Bahurmiz and Ng, 2007;

Miller et al., 2007).

In order to lessen the dependence on the fish oil there is a dire need to find out other

resources and to work out their nutritional efficacy so that reliance on fish oil may be reduced.

In present study an effort was therefore, made to evaluate the efficacy of different plant oil

sources. Plant oils stand out as the most likely candidates to partly substitute for fish oils in

fish feeds. Their total global production is around hundred times higher than that of fish oils

(Bimbo, 1990).

The Indian major carps, namely rohu Labeo rohita, catla Catla catla, mrigal

Cirrhinus mrigala are considered to be major aquaculture species in tropical countries,

contributing about 97% of the total fresh water aquaculture production. These are the most

commercially important freshwater fishes in India due to their relatively fast growth rate and

consumer preference. Together, these carps account for approximately 75% of the total inland

aquaculture production in India. C. mrigala the fish under study is a promising species for

aquaculture exploitation with its omnivorous feeding habits, rapid growth and good market

potential.

Although quantitative lipid requirements have been worked out for many fish species

including European eel, Anguilla Anguilla (Degani, 1986); red drum, Sciaenops ocellatus

(Daniels and Robinson, 1986; Ellis and Reigh, 1991; Buchet et al., 2000); gilthead seabream,

Sparus aurata (Marais and Kissil, 1979; Koven et al , 1992; Ibeas et al., 1994; Vergara et al.,

1999; Company et al.,1999; Seiliez et al., 2006); lobster, Homarus americanus (Kean et al.,

1985); Nile tilapia, Oreochromis niloticus (Hanley, 1991; De Silva et al., 1991); mud crab,

Scylla serrata (Sheen and Wu, 1999); common carp, Cyprinus carpio (Geurden et al., 1995);

sunshine bass, Morone chrysops x M. saxatilis (Webster et al., 1995); stinging catfish,

Heteropneustes fossilis O^nwar and Jafri, 1995^); catla (Mukhopadhyay and Rout, 1996);

walking catfish, Clarias batrachus (Anwar and Jafiri, 1995*'; Mukhopadhyay and Mishra,

1998); common dentex, Dentex dentex (Espinos et al., 2003); Atlantic salmon, Salmo salar

(Hillestad et al., 1998); giant fi-esh water prawn, Macrobrachium rosenbergii (Hari and Kurup,

2006); European seabass, Dicentrarchus labrax (Lanari et al., 1999; Peres and Teles, 1999);

abalone, Haliotis tuherculata x H. discus hannai (Mai et al 1995); greater amberjack, Seriola

dumerilli (Jover et al., 1999); cobia, Rachycentron canadum (Chou et al., 2001); turbot, Psetta

maxima (Regost et al., 2001; Kaushik, 2001); red porgy, Pagrus pagrus (Schuchardt et al.,

2008) korean rockfish, Sebastes schlegeli (Lee, 2001); haddock, Melanogrammus aeglefmus

(Nanton et al., 2001); spotted sorubim, Pseudoplatystoma coruscans (Martino et al., 2002;

2005); ayu, Plecoglossus altivelis (Lee et al., 2002); white sturgeon, Acipenser transmontonus

(Gawlicka et al., 2002); rock lobster, Jasus edwarsii (Johnston et al., 2003); prawn, Penaeus

indicus (Ali, 1990); Malabar grouper, Epinephelus malabaricus (Lin and Shiau, 2003);

pircanjuba, Brycon orbignyanus (Borba et al., 2006); rohu (Satpathy et al., 2003; Mishra and

Samantary, 2004); bastard halibut, Paralichthys olivaceus (Kim et al., 2006); Eurasian perch,

Perca fluviatilis (Kestemont et al., 2001); gibel carp, Carassius auratus gibelio and Chinese

longsnout catfish, Leiocassis longirostris (Pie et al., 2004); grey mullet, Mugil cephalus

(Argyropoulou et al., 1992); brown trout, Salmo trutta (Arzel et a l , 1994); bagrid catfish,

Pseudobagrus fulvidraco (Kim and Lee, 2005); senegalese sole, Solea senegalensis (Morals et

al., 2005); orange-spotted grouper, Epinephelus coioides (Luo et al., 2005; Zhi et al., 2005;

Lin, 2005); white seabass, Atractoscion nobilis (Lopez et al., 2006); turbot, Scophthalmus

maximus (Cho et al., 2005); haddock, Melanogrammus aeglefmus (Tibbetts et al., 2005); grass

carp, Ctenopharyngodon idella (Du et al., 2005; 2006); red snapper, Lutjanus campechanus

(Miller et al., 2005); yellowtail, Seriola quinqueradiata (Satoh et al., 2004); grouper,

Epinephelus coioides (vundu, Heterobranchus longifilis (Ovie et al., 2005); North Afi-ican

catfish, Clarias gariepinus (Ali and Jauncey, 2005); Siberian simgton,-Acipensor baeri

(Fontagne et al, 2006); pike-perch, Sander lucioperca (Zakes et al., 2004; Molnar et al., 2006);

white seabream, Diplodus sargus (Sa et ai., 2006); pircanjuba, Brycon orbignyanus (Borba et

al., 2003; 2006); green swordtails, Xiphophorus helleri (Ling et al., 2006); fresh water catfish,

Mystus montanus (Raj et al., 2007); ivory shell. Babylonia areolate (Zhou et al., 2007); pacu,

Piaractus mesopotamicus (Abimorad et al., 2007); Atlantic halibut, Hippoglossus hippoglossus

(Berge and Storebakken, 1991; Martins et al., 2007); Brazilian codling, Urophycis brasiliemis

(Bolasina et al , 2007); yellow croaker, Pseudosciaena crocea (Ai et al., 2008); cobia,

Rachycentron canadum (Niu et al., 2008) and Java barb, Puntius gonionotus (Mohanta et al.,

2008), very little information is available (Jafri et al., 1995; Marimuthu and Sukumaran, 2001)

on lipid requirement of the fish under study.

Substantial data have been generated on utilization of different lipid sources by a

number offish species like catia (Mukhopadhyay and Rout, 1996; Priya et al., 2005); turbot

(Adron et al., 1976; Leifson et al., 2003); chinook salmon, Oncorhynchus tschawytscha

(Mugrditchian et al., 1981; Welker et al., 2003; Huang et al , 2008); coho salmon, O. kisutch

(Dosanjh et al., 1984); mahseer, Tor Khudree (Bazaz and Keshavnath, 1993); rainbow trout

(Boggio, et al., 1985; Greene and Selivonchick, 1990; Caballero et al., 2002; Fonseca-Madrigal

et al., 2005; Bahrumiz and Wk, 2007); Atlanfic salmon (Thomassen and Rosjo, 1989; Polvi et

al., 1992; Dosanjh, 1998; Tocher et al., 2000; 2001; 2003; Torstensen et al., 2000; et al, 2001;

Grisdale-Helland et al , 2002; Bell et al , 2003; Bendiksen et al , 2003; Balfry et al, 2006;

Higgs et a l , 2006); broo.< trout, Salvelinus fontinalis (Guillou et a l , 1995); marron, Cherax

temimanus (Fotedar, 2004); sunshine bass (Nemaptipour and Gatlin, 1993); vundu

(Legendre et al., 1995); European sea bass (Yildiz and Sener, 1997; Izquierdo et al., 2003;

Montero et a l , 2005); bagrid catfish, Mystus nemurus (Ng et al , 2000; 2001); North Afiican

catfish (Lim et al , 2001; Olurin et al , 2004); red drum (Tucker et al., 1997); Japanese sea

perch, Lateolabrax japonicus (Du et al., 2002; Xue et al., 2006); Nile tilapia (Ali et al., 2000;

El-Sayed et al., 2005; Ochang et al., 2007); gilthead seabream (El-Kerdawy and Salama et al.,

1997; Aleman et al., 1998; Caballero et al., 2003; Izquierdo et al , 2003; 2005; Montero et al.,

2003; Martinez-Llorens et al., 2007); red seabream, Pagrus auratus (Gao and Lei, 1999;

Glencross et al., 2003); black seabream, Sparus macrocephalus (Ji, 1999); channel catfish,

Ictalurus punctatus (Gatlin and Stickney, 1972); brown trout (Truchini et al., 2003; 2005);

spotted sorubim (Martino et al., 2002); Murray cod, Maccullochellapeeliipeelii (Francis et al.,

2006; 2007 '*'); pike-perch (Molnar et al., 2006); grass shrimp, Penaeus monodon (Catacutan,

1991; Vasagam et al., 2005); largemouth bass, Micropterus salmoides (Subhadra ef al., 2006);

Atlantic cod, Gadus morhua (Morkore et al., 2007); grouper (Lin et al., 2007); shrimp,

Litopenaeus vannamei (Zhou et al., 2007) and grass carp ( Du et al., 2002; 2008) but no

information is available on the use of alternative lipid sources in the diet for fish under study.

Thus, the present study was, therefore, undertaken tc generate data on its lipid levels

and sources, and the findings are presented in the form of this dissertation which could be

used for developing the cost-effective practical feeds for the intensive culture of this species.

10

GENERAL INTRODUCTION

GENERAL METHODOLOGY

Source of fish stock and their acclimatization

Induced bred fingerling Cirrhinus mrigala were obtained from a G.B. Pant University of

Agriculture and Technology, Pantnagar. These were transported to the wet laboratory in

oxygen filled polythene bags, given the prophylactic dip in KMn04 solution (1:3000), and

stocked in rearing tanks (water volume 5000L) for a fortnight. During this period, the fish were

fed a mixture of soybean, mustard oil cake, rice bran and wheat bran in the form of moist cake

thrice a day at 0800, 1200 and 1700 hours. These were then acclimatized for one week on

casein-gelafin based (40 g/lOOg CP) H-440 diet (Halver, 2002).

Feeding ^pal

Fish of the desired size and number were sorted out from the acclimatized fish lots maintained

in the wet laboratory. These were stocked in triplicate groups in 70L high-density polyvinyl

circular troughs (water volume 55L) fitted with continuous water flow-through system. The

water exchange rate in each trough was maintained at 1.0-1.5 L/min. Fish were fed test diets in

the form of crumbles at 5% body weight twice daily at 0900 and 1600 h. The feeding trial lasted

for 8 weeks. Initial and weekly body weights were recorded on a top loading balance (Precisa

120A; 0.1 mg sensitivity, Oerlikon, AG, Zurich, Switzerland). Troughs were siphoned off to

11

remove faecal matter before feeding daily. Accumulation of the diet at the bottom of the trough

was avoided. Uneaten food was siphoned off immediately, dried in a hot air oven and

reweighed to measure the amount of food consumed. No feed was given on the day of weekly

measurement. Troughs were scrubbed and disinfected thoroughly with water and KMn04

solution on the day of weekly measurement. Mortality, if any, was recorded. At the end of the

experimental trial, desired number of fish were randomly sacrificed and kept in freezer (-20°C)

for the assessment of body composition.

Preparation of experimental diets

For studying the dietary lipid levels and sources of fingerling C. mrigala, test diets

were formulated with a different levels and sources of lipid. Calculated quantities of dry

ingredients were thoroughly stirred in a volume of hot water (80°C) in a steel bowl attached to

a Hobart electric mixer. Gelatin powder was dissolved separately in a volume of water with

constant heating and stirring and then transferred to the above mixture. Other dry ingredients

and oil premix, except carboxymethyl cellulose, were added to the lukewarm bowl one by one

with constant mixing at 40°C. Carboxymethyl cellulose was added last and the speed of the

blender was gradually increased as the diet started to harden. The final diet with bread dough

consistency was passed through a pelletizer fitted with 2-mm die to obtain pellets which were

dried in a hot-air oven at 40°C to reduce the moisture content below 10%. The dry pellets were

crumbled, sieved stored in a refrigerator.

Proximate analysis

Assessment of proximate composition of ingredients, diets and body was made using standard

12

techniques (AOAC, 1995). All the analyses were based on triplicate samples.'

Moisture

A known quantity of sample was taken in a pre-weighed crucible and placed in a hot air oven

at 105±rC for 24 hours. After complete drying, the sample was cooled at room temperature in

a desiccator and was revkcighed. The loss in weight gave an index of water from which its

percentage was calculated.

Ash

A known quantity of dried powdered sample (2-5g) was taken in pre-weighed silica crucible

and incinerated in a muffle furnace (S. M. Scientific Instrument (P) ltd. Jindal Company, India)

(600°C) for 2-4 hours or till the sample became carbon-free and completely white. The crucible

was cooled in a desiccator and reweighed to estimate the quantity of ash. The result was

expressed as percentage on dry weight basis.

Fat

Crude fat was estimated by continuous soxhiet extraction technique (Socs Plus, SCS 4, Pelican

equipments, Chennai, India) using petroleum ether (40-60°C B.P.) as solvent. Finely powered

and dried sample (2-4g) was placed in fat extraction thimble and placed in the soxhiet

apparatus. A clean, dry soxhiet receiver flask was weighed and fitted to the soxhiet assembly

on a boiling water bath for extraction, which was continued for 2-3 hours. After extraction the

flask was removed and kept in hot air oven (100°C) to evaporate the traces of solvent. It was

then transferred to a desiccator, cooled and reweighed. The difference between the weight of

13

the flask before and after gave the quantity of crude fat extracted from the unknown amount of

the sample. The result was expressed as percentage on dry weight basis.

Crude protein

The estimation was done using Kjeltec Tecator '' "' Foss, Hoeganaes Sweden. A known

quantity of sample was taken in Kjeltec digestion tubes. To this, 0.8 g of copper sulphate, 7.0 g

potassium sulphate and 12 ml. of concentrated sulphuric acid were added. The content was

digested in the digester of the instrument. The process of digestion continued for 30 minutes.

Now the digested sample was cooled at room temperature and titrated automatically in

distillation unit of the instrument. The level of protein displayed on the screen was noted down.

Gross energy

Gross energy was determined on a ballistic bomb calorimeter (Gallenkamp, Loughborough,

England). Prior to estimate, a knovm quantity of dried powdered sample (0.5-l.Og) was taken

in metallic crucible and compacted carefully to increase the rate of combustion at 25 lb oxygen

pressure. The heat generated upon combustion was read on the modulated galvanometer scale,

and converted to energy equivalent, worked out earlier using the thermo chemical grade

benzoic acid (6.32 kcal/g) as a standard. The gross energy was expressed as kcal/g. Energy of

ingredients used in the test diet was calculated as 5.52, 4.83, 3.83 and 9 kcal/g for casein,

gelatin, dextrin and fat, respectively as estimated on Gallenkamp ballistic bomb calorimeter.

Assessment of growth and conversion efficiencies

Calculations of the growth parameters were made according to the following formulae (Wee

and Tacon, 1982; Tabachek, 1986; Hardy, 1989; Gunasekera et al. 2000):

14

W -W Gain in live weight (%) = -^ ^ x 100

W,

Specific growth rate (%) = l ^ i ^ ^ ^ ^ J ^ i i ^ x 100

W2 = Final weight of fish

Wi = Initial weight of fish

D = Duration of the feeding trial (days)

„ , . . Dry weight of feed consumed Feed conversion ratio = —

Wet weight gain

Wet weight gain Protein efficiency ratio =

Protein consumed {dry weight basis)

Body protein deposition = Protein gain

Protein fed (TFxCP)

Protein gain = Final body protein x final body weight-Initial body protein x initial body

weight

TF = Total amount of diet consumed

CP = Percentage of crude protein in diet

STATISTICAL ANALYSIS

Responses of fingerling C. mrigala fed graded levels and sources of lipid were measured by

15

live weight gain percent (LWG %), feed conversion ratio (FCR), protein efficiency ratio

(PER), specific growth rate (SGR), body protein deposition (BPD) and by analyzing the body

composition. These response variables were subjected to one-way analysis of'variance

(ANOVA) (Snedecor and Cochran, 1968; Sokal and Rohlf, 1981). To determine significant

differences (P<0.05) among the treatment means, Duncan's Multiple Range Test (Duncan,

1955) was employed. Second-degree polynomial (Zeitoun et al. 1976) and broken-line

regression analysis (Robbins et al. 1979) was used to find out the optimum level in the growth

curve to predict more accurate response to the dietary intake. All the statistical analyses were

done using Matlab (version 6.5) and SPSS (version 13.0).

16

Table 1 Composition of mineral mixture"

Minerals (g/lOOg dry diet)

Calcium biphosphate 13.57

Calcium lactate 32.69

Ferric citrate 02.97

Magnesium sulphate 13.20

Potassium phosphate (Dibasic) 23.98

Sodium biphosphate 08.72

Sodium chloride 04.35

Aluminium chloride.6H2O 0.015

Potassium iodide 0.015

Cuprous chloride 0.010

Magnous sulphate. H2O 0.080

Cobalt chloride.6H20 0.100

Zinc sulphate 0.300

*Halver2002

17

Table 2 Composition of vitamin mixture''

Vitamins (g/lOOg dry diet)

Alpha cellulose 2.000

Choline chloride 0.500

Inositol 0.200

Ascorbic acid 0.100

Niacin 0.075

Calcium pantothenate 0.050

Riboflavin 0.020

Menadione 0.004

Pyridoxine HCl 0.005

Thiamin HCl 0.005

Folic acid 0.0015

Biotin 0.0005

Alpha tocopherol acetate** 0.040

Vitamin Bi2*** 0.00001 (0.5 ml)

*Halver 2002 **Incorporated with oil ***(10mg/500mlH2O)

18

• * • K m ,jt

Rearing tanks for the rearing of fry

H9r ji^inl

i ^^^^^^^^^^fc^^^^^H^B ^^^^1

^ i< -i^^n^^f^flff^^^B

Acclimatization of Indian major carp fry, Cirrhinus mrigala on experimental diet

Flow-through system used for conducting the feeding trials

Galienkamp ballistic bomb calorimeter used for the estimation of gross energy

Pelican equipments Socs Plus used for the estimation of fat

,2300 Kjeltec Tecator TM used for the estimation of proteins in the

sample

CHAPTER 1

CHAPTER 1

EFFECTS OF DIETARY LIPID LEVELS ON GROWTH, CONVERSION

EFFICIENCY AND BODY COMPOSITION OF FINGERLING MRIGAL,

CIRRHINUS MRIGALA (HAMILTON)

INTRODUCTION

For successful and sustainable culture of fishes nutritionally balanced, economically viable and

eco-friendly artificial feeds are warranted. Feed account for about 60% of the variable

operating cost, largely due to incorporation of higher percentage of protein. Generally, an

increase in the dietary protein up to certain levels improves growth but proportionally increases

feed cost. Excess protein in the feed is used for energy, not for growth when inadequate dietary

energy is fed. The expensive protein fraction should, therefore, be optimally utilized for growth

rather than for maintenance of fish (Chou and Shiau, 1996) and there should be a balance of

protein to energy in the feed (Ali et al., 2008). The reduction of protein level in the feed

formulation and increase in the non-protein energy soiu-ces to compensate the protein for

energy requirement not only increases the fish growth but also reduces the feed cost from

economic point of view (Phillips, 1969; Shiau and Lin, 2001; Ai et. al., 2004; Kim and Lee,

2005; Lopez et al., 2006, Ali et al., 2008).

Amongst non-protein energy sources in feed, lipid deserves special mention due to its

high calorific value than carbohydrates. Lipids are almost completely digestible by fish and

seem to be favored over carbohydrate as an energy source. Dietary lipids supply energy and

provide essential fatty acids needed for structural maintenance of membranes and proper

19

functioning of many physiological processes. Feeds with sufficient dietary lipids allow

maximal protein sparing and are relatively cost-effective (Garcia et al., 1981; Beamish and

Medland, 1986; Watanabe, et al., 1987; Sargent et al., 1989; Ellis and Reigh, 1991; Calduch-

Giner et al., 1999; Peres and Oliva-Teles, 1999; Fu et al., 2001; Skalli et al., 2004; Ai et al.,

2004; Ovie et al., 2005; Lee and Sang, 2005; Babalola and Apata, 2006; Fu and Cao, 2006;

Schulz et al., 2007, Raj et al. 2007; All et al. 2008). Taking into account the environmental

aspects, protein sparing by dietary lipids implies less discharge of nitrogen and phosphorous

compounds into the aquatic environment, providing more economic and environmentally

friendly diets (Davies et al., 1997; Hoffman et al., 1997; Medale et al., 1998; Carter and

Hauler, 2000; Hernandez et al., 2004). A number of studies have demonstrated that increasing

dietary lipids to an optimum concentration results in increased feed performance and protein

utilization by fish. There have been reports on improvements in feed conversion ratio (FCR)

and also nitrogen and phosphorus retention in fish when diets containing higher lipid levels

were provided (Hillestad et al., 1998; Hemre and Sandnes, 1999; Carter and Hauler, 2000;

Hernandez et al., 2004). On the other hand, high dietary lipid content might decrease feed

consumption and reduce growth (Watanabe, 1982). Moreover, high dietary lipid can also lead

to an increase in lipid deposition in fish body and affect quality and nutritional value of fish

meat. Therefore, the optimal dietary lipid level must be carefially evaluated and determined.

The quantitative lipid requirements have been worked out for many fish species and

reviewed under General Introduction section (page7-9).

Cirrhinus mrigala, the fish under study, is a potentially important freshwater fish

species cultured in Asia particularly in the Indian subcontinent as a component of polyculture

20

system. It is an excellent food fish with high market price and adaptability to intensive culture

systems. It has good acceptance to formulated feeds and relatively rapid growth rate. Its

protein, amino acids and energy requirements of fmgerling C. mrigala have been quantified

(Singh et al., 1987; 2008; Mohanty et al., 1990; Das and Ray,1991; De Silva and Gunasekera,

1991; Khan, 1991; Hassan et al., 1995; Marimuthu and Sukumaran, 2001; Benakappa and,

Varghese, 2004; Ahmed et al., 2003; 2004; Ahmed and Khan, 2004"''; 2005; 2006). However

very less information is available on lipid requirement of this fish species (Jafri et al., 1995;

Marimuthu and Sukumaran, 2001). Therefore, the present study was undertaken with a view to

generate data on the lipid requirement of fmgerling C. mrigala which could be used for

developing lipid balanced, cost-effective practical feeds for culture of this species on

commercial scale.

MATERIAL AND METHODS

Preparation of experimental diets

Casein-gelatin based isonitrogenous (40 g protein/lOOg dry diet) and isoenergetic (400

kcal/lOOg gross energy) diets with six levels of lipid (2, 4, 6, 8, 10 and 12 g/lOOg) were

formulated (Table 1). Levels of lipid were taken on the basis of the information available on

other fishes. The dietary protein level was fixed at 40 g/lOOg of the diet, reported optimum for

the growth of fingerling C. mrigala (Khan, 1991). Diets were made isoenergetic by adjusting

the amount of dextrin. A combination of cod liver oil and com oil (2:5) was used as a source of

lipid. Vitamin and mineral premixes were prepared as per Halver (2002).

Method of preparation of experimental diets has been discussed under general

21

methodology section (Page 12).

Experimental design and feeding trial

Source of the fish, their acclimation and details of the general experimental design has already

been discussed under the general methodology section (Pagel 1-12).

C. mrigala fingerling (4.2±0.20 cm; 0.62±0.02 g) were taken from the above

acclimated fish lot and stocked in triplicate groups in 70- L circular polyvinyl troughs (water

volume 55 L) fitted with a confinuous water flow-through (1-1.5 L min'') system at the rate of

20 fish per trough for each dietary treatment level. Fish were fed test diets in the form of

crumbles at 5% body weight twice daily at 0900 and 1600 h. Initial and weekly weights were

recorded on a top-loading balance (Precisa 120A; 0.1 mg sensitivity, Oerlikon AG, Zurich,

Switzerland). Fish were deprived of feed on the day they were weighed. The feeding trial

lasted for 8 weeks. Faecal matter, if any, was siphoned off before every feeding. Water quality

indices were monitored daily during the feeding trial and were recorded following standard

methods (APHA, 1992). The average water temperature, dissolved oxygen, free carbon

dioxide, pH, and total alkalinity based on daily measurements, were 26.5-29.3 °C, 6,5-7.1 mg

L"', 5.2-9.4 mg L"', 7.2-7.5 and 64.4- 81.6 mg L"', respectively.

Proximate analyses

Proximate composifion of casein, gelatin, experimental diets, and initial and final body was

estimated using standard methods as detailed earlier (12-14). Six sub samples of a pooled

sample of 40 fishes were analyzed for initial body composition. At the end of the experiment

10 fishes firom each replicate of dietary treatments were pooled separately. Six subsamples

22

from each pooled replicate were analyzed for final body composition.

Statistical analyses

Statistical analyses of growth data were done using procedures detailed earlier (15-16).

RESULTS

Effects of dietary lipid levels on growth parameters are presented in Table "2. Over the eight

week growth trial, significant differences (P<0.05) in live weight gain per cent of fingerling C.

mrigala fed diets containing graded levels of lipid were observed. Live weight gain% (LWG

%), protein efficiency ratio (PER), specific growth rate (SGR%), feed conversion ratio (FCR)

and body protein deposition (BPD) were significantly affected by dietary lipid levels. Fish

receiving 6g/100g dietary lipid (D3) reflected a maximum LWG% (428.9%), best PER (1.51),

SGR (2.97), FCR (1.66) and BPD (22.40) and significant fall was observed in fish fed diets

with more than above 6 g/lOOg dietary lipid level (D4-D6). Poorest LWG% (303.1%), PER

(0.83), SGR (2.48), FCR (2.99) and BPD (11.02) were observed in fish fed diet containing 12

g/lOOg lipid (D6).

In order to generate more precise data on lipid requirement of fingerling C. mrigala all

the growth data were subjected to second-degree polynomial regression and broken-line

regression analyses. On subjecting the live weight gain data to second-degree polynomial

regression analysis (Zeitoun et al., 1976), optimum level is estimated to be 5.8 g/lOOg of the

dry diet. (Fig.l). The relationship being;

Y=-2.825X^+32.8X+307.9 ( R V 8 5 9 )

23

The live weight gain to dietary concentrations of lipid relationship was estimated by the

following broken-line regression equation (Fig.l). A break point was evident at 5.25 g

lipid/1 OOg of the diet. The relationship being;

Y = 1 8 . 2 1 X + 3 2 0 . 4 ( R M . 9 9 8 ) , Y = - 1 6 . 2 5 X + 5 0 1 . 5 ( R M . 9 3 6 )

The FCR of C. mrigala fingerling fed 6 g/lOOg lipid diet differed significantly (P<0.05)

from the other levels of dietary lipid inclusion. The FCR (Y) to dietary concentrations of lipid

(X) relationship was estimated by the following second-degree polynomial regression equation

(Fig. 2).The optimum lipid level is estimated to be 6.03 g/lOOg of the diet. The relationship

being;

Y=0.02929X^-.3534X+3.054(R^=.803)

The FCR to dietary concentrations of lipid relationship was estimated by the broken-line

regression equation (Fig.2). A break-point was evident at 5.4 g lipid/1 OOg of the diet. The

relationship being;

Y = - . 1 9 7 5 X + 2 . 9 1 3 ( R M . 9 ] 8 ) , Y = 0 . 1 6 7 5 X + . 9 1 5 ( R M . 899)

The SGR (Y) to dietary lipid levels (X) relationship was described by the second-

degree polynomial regression analysis (Fig. 3). The optimum level is estimated to be 5.78

g/lOOg. The relationship being;

Y=-.011X^+.127X+2.518(R^=.901)

The SGR to dietary concentrations of lipid relationship was estimated by the broken-

24

line regression equation (Fig. 3). A break-point was evident at 5.56 g lipid/1 OOg of the diet.

The relationship being;

Y=0.065X+2.S83(R^=0.998),Y=-0.07X+3.33(R^=0.973)

The PER (Y) to dietary concentrations of lipid(X) relationship was estimated by the

following second-degree polynomial regression equation (Fig. 4).The optimum lipid level is

estimated to be 6.17 g/lOOg of the lipid. The relationship being;

Y=-.01442X^+.1779X+.723(R^=.643)

The PER to dietary concentrations of lipid relationship was estimated by the broken-line

regression equation (Fig. 4). A break-point was evident at 4.7 g lipid/1 OOg of the diet. The

relationship being;

Y=0.1225X+.7233(R^=0.882),Y=-0.0625X+1.598(R^=0.939)

Similarly, the BPD (Y) to dietary lipid levels (X) relationship was described by the

following second-degree jiolynomial regression equation (Fig. 5).The optimimi lipid level is

estimated to be S.lg/lOOg of protein. The relationship being;

Y=-.223X2+2.617X+10.76 (R^=.655)

The BPD to dietary concentrations of lipid relationship was estimated by the broken-line

regression equation (Fig. 5). A break point was evident at 4,56 g lipid/1 OOg of the diet. The

relationship being;

Y = 1 . 8 6 2 X + 1 0 . 4 2 ( R ^ = 0 . 8 7 6 ) , Y = - 1 . 0 5 5 X + 2 3 . 6 9 ( R M . 9 9 9 )

25

On the basis of the above second-degree polynomial regression and broken-line

analysis, maximum live v eight gain, best FCR, highest SGR, PER and BPD occurred in the

range of 4.56g/100g - 6.17 g/100 g of the dietary lipid.

Data related to body composition are summarized in Table 3. Body composition of

fmgerling C. mrigala was significantly (P<0.05) affected by the dietary lipid levels (Table 3).

Moisture content was negatively correlated to lipid content. The moisture content significantly

decreased with increasing dietary lipid levels. The amount of body lipid was found to be

positively correlated to the dietary lipid, the highest and lowest body lipid concentrations were

observed in fish fed diets D6 and Dl with 12 g/lOOg and 2 g/lOOg diet, respectively. Maximum

body protein was recorded in fish fed diet containing 6 g lipid/1 OOg diet (D3). Fish fed diets

(D4, D5, D6) containing more than 6 g lipid/1 OOg diet did not show any improvement in its

body protein content.

DISCUSSION

In fish nutrition for the provision of energy and essential fatty acids lipids play an important

role. Dietary lipids are carriers of fat-soluble vitamins and provide other compounds such as

polar h'pid which are important for structural components of the cell membrane. Several studies

have shown that providing adequate energy with dietary lipids can minimize the use of more

high-priced protein as an energy source (Ai et al., 2004; Hung et al., 2004; Kim and Lee, 2005;

Lopez et al , 2006). Increasing lipid levels of fish feeds has showi to be an effective approach

to improving feed efficiency, protein utilization and decreasing nitrogen waste output (Carter

and Hauler, 2000; Hillestad et al., 1998; Hemre and Sandness, 1999; Hernandez et al, 2004).

Moreover, the nutritional strategy for protein sparing effect is to increase adequate amount of

26

lipid in fish diet to reduce protein inclusion without compromising growth (Sargent et al.,

2002; Ai et al., 2004; Lopez et al., 2006).

In this study, effects of increasing dietary lipid levels were observed on growth, SGR,

FCR and PER of fmgerling C. mrigala. The growth response data indicated that the maximum

growth was obtained at 6 g/lOOg dietary lipid. Earlier studies on species like channel catfish,

Ictalurus punctatus (Dupree, 1969; Stickney, 1984); rainbow trout, Salmo gairdneri (Watanabe

et al., 1979; Reinitz and Hitzel, 1980); red drum, Sciaemps ocellatus (William and Robinson,

1988; Ellis and Reigh, 1991; Serrano et al., 1992); stinging catfish, Heterppnuestes fossilis

(Anwar and Jafri, 1995); grouper, Epinephelus coioides (Shiau and Lan, 1996; Lin and Shiau,

2003; Luo et al., 2005); grass carp, Ctenopharyngodon idella (Du et al., 2005); haddock,

Melanogrammus aeglefinus (Tibbetts et al., 2005);fresh water catfish, Mystus montanus (Raj et

al., 2007) prawn, Macrobrachium rosenbergii (Hari cJid Kurup, 2006); ivory shell, Babylonia

areolate (Zhou et al., 2007); silver barb, Puntius gonionotui, (Mohanta et al., 2008) indicated

that incorporation of 5-12 g/lOOg dietary lipid is optimal for growth. Supplementation of extra

lipid to the diet did not improve LWG%, SGR and PER in C. mrigala. Similar results were

also evident in several earlier studies (Dupree, 1969; Andrews et al., 1978;, Anwar and Jafri,

1995; Weatherrup et a l , 1997; Silverstein et al., 1999; Regost et al., 2001; Espinos et al., 2003;

Pie et al., 2004; Lee and Sang, 2005; Du et al., 2005; Lopez et al., 2006 and Mohanta et al.,

2008). The growth reduction at high lipid level could be due to the limited ability to digest and

absorb high amounts of lipid, a reduction in feed intake, excess lipid accumulation in liver and

other visceral organs or creation of dietary or metabolic imbalances (Luo et al., 2005).

The SGR increased from 2.48-2.97% with increase in dietary lipid from 2-6 g/lOOg diet

27

(D1-D3) and thereafter decreased in present study. The trend of increasing SGR with increase

in dietary lipid up to above level and then no improvement in SGR with further rise in dietary

lipid level (D4-D6) is in agreement with the earlier workers in Tilapia aurea (Stickney and

McGeachin, 1983); stinging catfish (Akand et al., 1991; Anwar and Jafri, 1995);walking

catfish, Clarias batrachus (Anwar and Jafri, 1995); dentex, Dentex dentex (Espinos et al.,

2003); grass carp (Du et al., 2005); white seabass, Atractoscion nobllis (Lopez et al., 2006);

ivory shell (Zhou et al., 2007); fresh water catfish (Raj et al., 2007); silver barb (Mohanta et

al., 2008).

The positive correlation between body lipid content and dietary lipid (table 3) indicates

that when dietary lipid is supplied in excess, a proportion of this lipid is deposited as body fat.

This is in agreement with the results reported on other fish species such as rainbow trout,

Onchorynchus mykiss (Lee and Putnam, 1973; Rasmussen et al., 2000); channel catfish

(Garling and Wilson, 1977), common carp (Takeuchi et al., 1979); red drum (Ellis and Reigh,

1991); hybrid catfish, Clarias macrocephalus x Clarias gariepinus (Jantrarotai et al., 1994);

abalone, Haliotis tuberculata and H. discus hannai (Mai et al., 1995); haddock (Nanton et al.,

2001); surubim, Pseudoplatystoma coruscans (Martino et al., 2002); rohu, Labeo rohita

(Satpathy et al., 2003), grouper (Zhi et al, 2005), Chinese longsnout catfish, Leiocassis

longirostris (Tan et al., 2007), silver barb (Mohanta et al., 2008). A significant reduction in

protein content was noted at higher dietary lipid levels that may be due to inefficient utilization

of protein at higher energy levels (D4, D5 and D6). The same results were observed in case of

other fish species such as walking catfish, (Anwar and Jafri, 1995), tilapia, 0. niloticus x 0.

aureus (Chou and Shiau, 1996); cobia, R. canadum (Wang et al., 2005); white seabass (Lopez

et al., 2006); ivory shell (Zhou et al., 2007), fresh water catfish (Raj et al., 2007).

28

On the basis of the second-degree polynomial and broken-line regression analyses of

LWG%, FCR, SGR, PER and BPD data it is concluded that dietary lipid in the range of 4.56-

6.17 g/lOOg is appropriate for maximum growth and efficient feed utilization.

SUMMARY

An eight week feeding trial was conducted to evaluate the effects of graded dietary lipid levels

on grow^, conversion efficiencies and body composition of fingerling Cirrhinus mrigala

(4.2±0.20 cm; 0.62±0.02 g). Casein-gelatin based isonitrogenous (40 g protein/1 OOg diet) and

isocaloric (400 kcal/lOOg gross energy) purified diets with graded levels of lipid (2, 4, 6, 8, 10

and 12 g/lOOg dry diet) were fed to triplicate groups of fishes at the rate of 5% body weight at

0900 and 1600h. Maximum live weight gain (LWG%) (428.9%), protein efficiency ratio

(PER) (1.51), specific growth rate SGR (2.97), best feed conversion ratio (FCR) (1.66) and

body protein deposition (BPD) (22.40) were recorded in fish fed diets with 6 g lipid/1 OOg diet

(D3). Fish fed diets with 12 g lipid/lOOg diet (D6) reticcted significantly lower LWG

(303.1%), FCR (2.99), PER (0.83), SGR (2.48) and BPD (11.02) among the other treatments.

Maximum body protein and body protein deposition was found at 6 g/lOOg dietary lipid

inclusion. LWG%, FCR, PER, SGR and BPD improved (P<0.05) as dietary lipid level

increased from 2 to 6 g/lOOg (D1-D3) of the diet whereas at still higher levels growth

responses were reduced (P>0.05), indicating that 6 g lipid/1 OOg diet satisfied the requirement.

Hence, it is recommended that an inclusion of 6 g lipid/1 OOg in 40 g protein/1 OOg diet is

optimum for developing practical feeds for the mass rearing of fingerling C. mrigala.

29

Table 1 Composition of experimental diets

Ingredients

(g /lOOg dry diet)

Casein 1

Gelatin^

Dextrin

Com oil

Cod liver oil

Mineral mix ' . 3.5

Vitamin mix a- Cellulose

Carboxymethyl cellulose

Total

Crude protein

Analyzed crude protein

Total lipid

Analyzed lipid

Gross energy^ (kcal-lOOg"')

(Dl)2

40

10

29.48

1.43

0.57

4

3

6.50

5

100

40

40.5

2

2.1

400

Dietary lipid

(D2)4

40

10

24.78

2.85

1.14

4

3

9.22

5

100

40

39.85

4

4.03

400

(D3)6

40

10

20.08

4.28

1.71

4

3

11.92

5

100

40

39.69

6

6.1

400

levels (g/lOOg)

(D4)8

40

10

15.38

5.72

2.28

4

3

14.62

5

100

40

40.8

8

8.25

400

(D5) 10

40

10

10.68

7.14

2.85

4

3

17.32

5

100

40

40.1

10

9.98

400

(D6) 12

40

10

5.98

8.57

3.42

4

3

20.02

5

100

40

40.01

12

12.37

400

Crude Protein (76%); Crude Protein (97%); ^Halver (2002); "Mineral mixture (glOOg"') calcium biphosphate 13.57; calcium lactate 32.69; ferric citrate 02.97; magnesium sulphate 13.20; potassium phosphate (dibasic) 23.98; sodium biphosphate 08.72; sodium chloride 04.35; aluminium chloride.6H20 0.0154; potassium iodide 0.015; cuprous chloride 0.010; magnus sulphate. HjO 0.080; cobalt chloride. 6H2O 0.100; zinc sulphate. 7H2O 0.40; 'Vitamin mixture (Ig vitamin mix +2g oe-cellulose) choline chloride 0.500; inositol 0.200; ascorbic acid 0.100; niacin 0.075; calcium pantothenate 0.05; riboflavin 0.02; menadione 0.004; pyridoxine hydrochloride 0.005; thiamin hydrochloride 0.005; folic acid 0.0015; biotin 0.0005; alpha-tocopherol 0.04; vitamin 6,2 0.00001; Loba Chemie, India "^Calculated on the basis of fuel values 23.08, 20.199, 16.02 and 37.64 Id for casein, gelatin, dextrin, and fat, respectively, as estimated on Gallenkamp ballistic bomb calorimeter.

30

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o d +1 m 0 0

•a <

m - -

CN

d +1 CN

0 0 o d +1 T f CN CN

CN O d +1 CN

NO

C3N O

i n ON

^ ;t

o

CO O &, u

T3

2 O H

o

o o A

•a

2

V3 a

o -H

U

•.3 U

O ^

> >

ss

(N en

Fig. 1 Second-degree polynomial and broken-line relationship of

dietary lipid levels to live weight gain%

500

480

460

440

"T T -1 r

a.<\

• J ^ " i . " " ^ ^ '

<»v

^•^Y=-Z825X2+32.8X+307.9(R2=0.8S91

3 4 5 6 7 8 9

Dietary lipid levels (g/lOOg dry diet)

Fig.l

34

Fig. 2. Second-degree polynomial and broken-line relationship of

dietary lipid levels to feed conversion ratio

U G _o

U

a o u -a V V to

3

2.8

2.6

2.4*

2.2

2

1.8

1.6

1.4

1.2

1

T 1 1 1 1 1 1 1 I ; n

^ / '

, - ' . ' - '

,* . ' '

5.4g/100gm 1 1 1 1

Y=0.02929X2-.3534X+3.054(R2=0.803)

1

X " s . >. •V.

6.03q/100gm " x,

3 4 5 6 7 8 9 10 11 12

Dietary lipid levels (g/lOOg dry diet)

Fig. 2

36

Fig. 3 Second-degree polynomial and broken-line relationship of

dietary lipid levels to specific growth rate%

3.2

3.1

3

J 2.9 k.

J3 ^ 2.8 o u y 2.7'

c '3 a> a 2.6

CZ5

2.5

1 1 1 1 1 I 1 1 \~^ 1 T

, - ' T '

^^J^^^^

.^"^^

5.56g/100gm! 1 1 1 1 i

*x .iJli.^(^.011X2+0.127X+2.518(R2=0.901)

': * ' ^ ^ ^ ^ " " ^ " ^ N ^ •^. ^ ^ X s .

V »

j 5.78g/100gm N X x 1 1 1 1 1

2 3 4 5 6 7 8 9 10 11 12

Dietary lipid levels(g/100g dry diet)

Fig. 3

38

Fig. 4 Second-degree polynomial and broken-line relationship of

dietary lipid levels to protein efficiency ratio

1.8

1.6

-[ 1 : r—I r

'^•-. .""' Y=-0.01442X2+0.1779X+0.723(R2=0.643) - . _,—

6.17g/10C^m

2 3 4 5 6 7 8 9 10 11 12

Dietary lipid levels(g/100g dry diet)

Fig. 4

40

a

O eu

a

•«< o a

o

28

26

24

22

20

18

16

14

12

10

1 • 1 1

» " - - v ^

^^^ . .^

1 •• 1 1 1 1 ^ 1 1

#» f ^ x '

. < » •

^'^^^H Y=-.223X2+2.617X+10.76 (R2=.655)

^ ^ r

4.56g/100gm 1 1

•^ ' S ' ' ' ' * ^ ^^^ .^ * > . ^ ^ ^ K ^

• ^ * ^ ^ v ^

j 5.1g/100gm — u i . 1 1 1 1 1

1

-

-

_

-

\ ^

1

2 3 4 5 6 7 8 9 10 11

Dietary lipid levels (g/lOOg dry diet) 12

Fig. 5

42

CHAPTER 2

CHAPTER 2

EFFECTS OF DIFFERENT LIPID SOURCES ON GROWTH, CONVERSION

EFFICIENCY AND BODY COMPOSITION OF FINGERLING MRIGAL,

CIRRHINUS MRIGALA (HAMILTON)

INTRODUCTION

Fish oil is a major dietary lipid source used in fish feeds because of its high proportion of

long-chain n-3 fatty acids, which are nutritionally essential to teleosts (NRG, 1993).

Other than providing a source of energy and essential fatty acids, it is commonly used to

coat the extruded pellets to improve the palatability and appearance of the feed. The

stagnation in global fish oil production coupled with increased demand for its use in

aquaculture feeds, especially, has greatly inflated fish oil prices (Barlow, 2000). Because

of predictable insufficient fish oil availability for fish feed and in order to sustain the

rapid grov^ of the aquaculture, there is need to partially or totally replace fish oil with

cheaper and sustainable sources of dietary lipid. So, there is currently great interest to

reduce feed costs by using locally available or alternative lipid sources.

Vegetable oils are the obvious candidates to replace fish oil and the global

production of the main seed oils has increased in recent years so that the price and

availability of vegetable oil have been relatively constant. Successful replacement of fish

oil by vegetable oil would reduce both the complete dependence on fish oil as a raw

material and its related cost (Palmegiano et al., 2008).Vegetable oils are potential lipid

sources, as they are virtually free of dioxins and other organic pollutants and can

43

maintain the demand for fisli oil at sustainable levels (Figueiredo-silva et al., 2005). Most

of the vegetable oils are rich in fatty acids with 18 carbons, and many of them are also

rich in linoleic or linolenic acids, essential for freshwater fish species. Therefore, many

vegetable oils are a good energy source in diets for freshwater species such as vundu,

Heterobranchus longifilis (Legendre et al., 1995); common carp, Cyprinus carpio

(Fontagne et al., 1999); catla, Catla catla (Mukhopadhyay and Rout, 1996; Priya et al.,

2005); grass carp, Ctenopharyngodon idella (Du et al., 2002; 2008).

Culture of Indian major carps namely rohu, Labeo rohita, catla, Catla catla,

mrigal, Cirrhinus mrigala contribute substantially to the inland production. C. mrigala,

the fish under study normally attains 800-1000 g in the a year, depending on stocking

density and management practices (Jhingran and Pullin 1988).

A number of studies have been conducted on utilization of different lipid sources

on many fish species and reviewed under General Introduction section (page 9-10), no

information is available on the effect of different lipid sources on growth offish under

study. Thus, the present study was conducted to evaluate the effects of growth conversion

efficiencies and body composition of different dietary lipid sources of fingerling C.

mrigala.

MATERIALS AND METHODS

Experimental diets

Six isonitrogenous (40% CP) and isoenergetic (400 kcal-100 g''GE) test diets Dl

(Cod-liver oil); D2 (Corn oil); D3 (Mustard oil); D4 (Sunflower oil); D5 (Soybean oil)

44

and D6 (Coconut oil) were prepared with different lipid sources. The dietary protein level

was fixed at 40 g/lOOg CP reported optimum for growth of Cirrhinus mrigala (Khan,

1991). Based on the results obtained in experiment 1, dietary lipid level was fixed at 6

g/lOOg of the dry diet. Diets were made isoenergetic by adjusting the amount of dextrin.

Preparation of experimental diets

Method of preparation of experimental diets has been discussed under general

methodology section (Page 12).

Experimental design and feeding trial

Source of the fish, their acclimation and details of the general experimental design have

already been discussed under the general methodology section (Page 11-12).

C. mrigala, fingerling (4.21 ±0.05 cm; 0.61 ±0.03 g) were stocked randomly in

triplicate groups in 70-L circular polyvinyl troughs (water volume 55L) fitted with a

continuous flow-through system at the rate of 20 fish per troughs for each dietary

treatment levels. The fish were fed experimental diets in the form of crumbles at the rate

of 5% body weight at 0900, and 1600h. No feed was offered to the fish on the day they

were weighed. The feeding trials lasted for eight weeks. Initial and weekly body weights

were recorded on a top loading balance (Precisa 120A; AG, Oeriikon, Zurich,

Switzeriand). Troughs were siphoned off to remove faecal matter before feeding daily.

Any uneaten feed was siphoned off immediately, dried in a hot air oven and reweighed to

measure the amount of feed consumed. Water quality parameters were recorded daily

during the feeding trial (APHA, 1992). The average water temperature, dissolved oxygen.

45

free carbon dioxide, pH and total alkalinity based on daily measurements were 26.8-27.9

°C, 6.7-7.1 mg L"', 5.5-10.7 mg L"', 7.5-7.8 and 65.7-80.5 mg L'', respectively.

Proximate analyses

Proximate composition of casein, gelatin, experimental diets, and initial and final body

was estimated using standard methods as detailed earlier (12-14). Six sub samples of a

pooled sample of 40 fishes were analyzed for initial body composition. At the end of the

experiment 10 fishes from each replicate of dietary treatments were pooled separately.

Six subsamples from each pooled replicate were analyzed for final body composition.

Statistical analyses

Statistical analyses of growth data were done using procedures detailed earlier (15-16).

RESULTS

Over the 8-week: growth trial, the growth rates and feed utilization efficiencies of C.

mrigala fingeriings fed diets with varying sources of lipid were not significantly different

(P>0.05). At the end of the feeding trial, C. mrigala attained a live weight gain per cent

(LWG%), feed conversion ratio(FCR), specific growth rate(SGR) and protein efficiency

ratio(PER) in the range of 458-479%, 1.49-1.57, 3.13-3.07 and 1.68-1.59 respectively.

The whole-body proximate composition was also not affected by different lipid sources.

Moisture content averaged about 79.43%. Whole-body lipid and the protein content

ranged from 3.1% to 3.17% and 12.01% to 13.68% respectively.

46

DISCUSSION

Lipid plays an important role in fish nutrition as a source of energy, essential fatty acids

(EFA) and other lipid classes, such as phospholipids, sterols and fat-soluble vitamins.

Among the various lipid sources, fish oil is generally used as a dietary lipid source due to

the richness of essential n-3 fatty acids and fat-soluble vitamins. But, consistently higher

prices are anticipated for fish oil. This, in turn, has necessitated the search for alternatives

to ensure the fiiture profitability of aquaculture (Higgs et al., 2006). In comparison, the

global production of A'egetable oils has increased in recent years reaching volumes

hundred times that of fish oil. The price of vegetable oils have been more constant and

even decreasing in some markets. Therefore, use of vegetable oils in fish diets would

reduce both the absolute dependence on fish oil and associated costs. (Izquierdo, 2003).

In the present study, many alternative sources of plant lipid have been tested for

their suitability in the diet of mrigal. All the experimental diets were accepted well by

mrigal because dietary protein and lipid meet this species requirement. No significant

differences in growth were found between fish fed the fish oil and different vegetable oil

diets. Similar reports have been' demonstrated for brook charr, Salvelinus fontinalis

(Guillou et al., 1995); Atlantic salmon, Salmo salar (Thomassen and.Rosjo, 1989;

Rosenlund et al., 2001; Bell et al., 2002); surubim, Pseudoplatystoma coruscans (Martino

et al., 2002), turbot, Psetta maxima (Regost et al., 2003); gilthead seabream Sparus

aurata (Izquierdo et al., 2003); barramundi, Lates calcarifer (Raso and Anderson, 2003)

and grouper, Epinephelus coioides (Lin et al., 2007).

47

No significant differences in FCR and PER were also found in fish fed the fish oil

diet compared with those fed vegetable oil diets. These results were similar to those

found by several other authors (Martino et al., 2002; Millamena, 2002; Izquierdo et al,

2003; Raso and Anderson, 2003; Molnar et al., 2006; Lin et al., 2007). No significant

differences in growth and conversion efficiencies of fish fed diet containing fish oil and

vegetables oils and indicate that all lipid sources were efficiently utilized by the fish and

the fatty acid profile of all oils was probably as per as the need of C mrigala and the

added various lipid sources were used for energy.

Vegetables oil may be alternatives to fishmeal diets for C. mrigala fingerling. No

significant differences in the growth performance and feed utilization of fish fed diets

with vegetables oil and that of fish oils, suggest reduced dependence on fish oil.

However, Jordal et al. (2007) pointed out that high dietary vegetable oil inclusion

increases hepatic triacylglycerol stores and decreases plasma lipid levels.

During the present study, no significant differences were observed in the whole

body composition of C. mrigala. Similarly no significant differences in body composition

offish fed diets with different lipid sources were reported in the past (Mugrditchian et al.,

1981; Hardy et al., 1987; Martino et al., 2002; Ng et al., 2003; Regost et al, 2003; Raso

and Anderson, 2003; Molnar et al., 2006; Lin et al, 2007; Stubhaug et al., 2007).

However, lipid contents of whole body were significantly different amongst the different

dietary treatments reported by several workers (Catacutan 1991; Lim et al., 1997

Gonzalez-Felix et al., 2003; Vasagam et al. 2005; Zhou et al., 2007).

48

The results of the present study indicate that various types of plant oils in place if

fish oils can be used in diets fingerling C. mrigala without affecting growth, conversion

efficiencies and conversion efficiencies.

SUMMARY

The aim of this study was to determine the feasibility of using vegetable oils as

alternative sources and assess the effects on growth conversion efficiencies and body

composition of fingerling C. mrigala (4.21 ±0.05 cm; 0.61 ±0.03 g). Six casein-gelatin

based isonitrogenous (40 g protein/1 OOg diet) and isolipidic (6 g lipid/1 OOg diet) diets

were formulated containing fish oil (Dl), corn oil (D2), mustard oil (D3), soybean oil

(D4), sunflower oil (D5) and coconut oil (D6). Crumbled diets were fed to triplicate

groups of fishes at the rate of 5% body weight at 0900 and 1600h. No significant

differences in live weight gain, specific growth rate, feed conversion ratio and protein

efficiency ratio were recorded between fish fed diet containing fish oil (Dl) and

vegetable oils diets (D2, D3, D4, D5, D6). Dietary lipid sources did not affect whole

body composition among the various diets. The present results suggest that alternative

vegetable lipid sources can be used without affecting growth and feed utilization

efficiencies.

49

Table 1 Composition of experimental diets used for evaluation of different lipid sources in

diet for fingerling C. mrigala.

Dietary lipid sources

Ingredients (g/lOOg) dry diet

(DI) (D2) (D3) (D4) (D5) (D6)

Casein'

Gelatin^

Dextrin

Cod liver oil

Com oil

Mustard oil

Sunflower oil

Soybean oil

Coconut oil

Mineral mix

Vitamin mix"*

a- Cellulose

Carboxymethyl cellulose

Total

Total protein Analyzed protein Total lipid Analyzed lipid Gross energy^ (kcal/lOOg)

40

10

20.1

6

40

10

20.1

.

40

10

20.1

.

40

10

20.1

-

40

10

20.1

.

40

10

20.1

.

4

3

11.9

5

100

4

3

n.9 5

100

4

3

n.9 5

100

4

3

n.9 5

100

4

3

n.9 5

100

40 40 41.02 40.2 6 6 6.02 5.99 400 400

6

4

3

n.9 5

100

40 40 40 40 40.12 40.05 39.92 40.01 6 6 6 6 6.12 6.05 6.09 6 400 400 400 400

'Crude Protein (76%); ^Cnide Protein (96%); Halver (2002); 'Mineral mi>:ure (g/lOOg"') calcium biphosphate 13.57; calcium lactate 32.69; feme citrate 02.97; magnesium sulphate 13.20; potassium phosphate (dibasic) 23.98; sodium biphosphate 08.72; sodium chloride 04.35; aluminium chloride.6HjO 0.0154; potassium iodide 0.015; cuprous chloride 0.010; magnus sulphate. HjO 0.080; cobalt chloride. 6HjO 0.100; zinc sulphate. 7HjO 0.40; 'Vitamin mixture (Ig vitamin mix +2g ce-cellulose) choline chloride 0.500; inositol 0.200; ascorbic acid 0.100; niacin 0.075; calcium pantothenate OM; ciboflaviiv 0.02; menadione 0.004; pytidoxine hydvochtoiide 0.005; thiamin hydrochloride 0.005; folic acid 0.0015- biotin 0.0005; alpha-tocopherol 0.04; vitamin B,i 0.00001; Loba Chemie, India 'Calculated on the basis of fuel values 5.52,4.83, 3.83 and 8.29 kcal for casein, gelatin, dextrin and fat, respectively, as estimated on Gallenkamp ballistic bomb calorimeter.'

50

u s o

'a

i I i DD

u b£ S c o 9i

• wm <J

o *s 15 « e o (X U u >• e o

- a e es

o

Ji A H

V5

W

s o

' a

5

p.

Q

+1 o

+1

o

+1 o o

(N

+1

O

+1

o

bO

m o +1

O

+1

(N O +1

o +1 m '^

(N

+1

0 0

^

+1

+1

VO

+1 ON

00

rn

+1 00

(N O

+1 ^ o

(N O

+1

m'

0 0

+1

'^

+1

0 0 IT)

X

"S ^ rt

•^>> • f l

<u 00 l-c (U > <

•4-»

13

(U 00 iH

u > <

" ^ —'

00

op *QJ ^ u >

• J

US O

+1 ON

, -^ CO

o +1 IT)

.c i r i o o +1 m

o o +1 in

o +1 r--

u

+1 00 NO

O o +1 NO

O +1 CO NO

o o +1

o o +1 ON

o c

' o

'S o

+1 en

^H r-H r<1

O O +1 o

-^ — m

(N O o +1

>—I .—I m

o o +1

O

o" +1 NO NO

o o +1

1

1 ^^ -^ m

00 o O +1 ON

o •^ •^ en

O

+1 o

^^ ^-, fT)

2 (20 O

'o ex

00

e •5

5

p

o o

o d Z

C c __

itlilt litl

LUIII

'r 5 Q

(N O +1

' O^

>o *— o +1 oo VO

TT

o o +1 t--

lO

o o +1 00 <N

1

V U

O

an S O X

C M

t

DC

u

C

s o

o

o

o CQ

^ R

H

9 O

'S.

ID

Q

Q

J3

o +1 «f

ON l>

'—', O +1

rn

o o +1

*-^s

o +1 -*

o +1

o +1 in

ON

o +1

m o +1 o o 00

^ '^

O +1

c^ -:

+1

O +1

O O +1 O N IT)

o +1 o

O o +1

o +1

in

p o +1

--H m

p O +1

o +1 "* O N

o

+1

rn

o +1 O N

• ^ ' ^ - - "

o +1 O N <N

O +1 00

o +1

en

O o +1 O N

O"^

0)

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'o

55^

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—

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o o A 0-

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(N

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