m^sittx of $i)iio!^qpi)p - connecting repositories · imtiaz ahmed. the work is original and...

DIETARY AMIIVIO ACID REQUIREMENT OF SELECTED TELEOSTEAIM FISH SPECIES

>/ DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF THE DEGREE OF

M^sittx of $i)iIo!^QpI)p • ^ . f f IN

ZaOLOGY

BY

IMTIAZ AHMED

FISH NUTRITION RESEARCH LABORATORY DEPARTMENT OF ZOOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2001

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DS3205

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Phones I Ex'efnal : 700920/21-300/301 ( Internal : 300/301

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

ALIGARH—202002

INDIA

Sections :

1. AGRICULTURAL NEMATOLOGY D. No ...../?,D ., 2. ENTOMOLOGY O , n o ,-, n, 3. FISHERY SCIENCE &AQUACULTURE Dated f;.l • ' ^ • i-^ 0 / . . 4. GENETICS •"•" 5. PARASITOLOGY

This is to certify that the Dissertation entitled "Dietary amino acid

requirement of selected teleostean fish species" has been completed

under my supervision by IVIr. Imtiaz Ahmed. The work is original and

independently pursued by the candidate. 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.

Dr. Mukhtar A. Khan

CONTENTS

ACKNOWLEDGEMENTS

GENERAL INTRODUCTION

Page No

GENERAL METHODOLOGY 10

CHAPTERS

1 Dietaiy threonine requirement of fingerling Indian major carp, 17 Cirrhimis mrii^ala (Hamilton).

2 Dietaiy methionine requirement of fmgerling Indian major caip, 25 Ciirhiniis mri^ala (Hamilton).

REFERENCMvS 36

Acl(nowCedgements

I fed extreme[y ftappy to express my sincere gratitude to my researcft guide, <Dr. ^ul{fitarj^. Xfum, for fii'i invafua6(e suggestions, constructive criticism and encouragement.

I am tHan^uC to (prof fl.%, Jafri, Cft-ciirman, (Department of Zoofogy for providing necessary (aSoratory facilities andinvaCuxiSk suggestions.

I aCso tal{e tfiis privilege to express my fteartfuC tfianf{s to my teacHers, (prof Jisfjl. T(Jtan, (Dr 5\i. Sftafiid Siddiqui, (Dr Sardar Mafimood 'Kfian, <Dr Iq6aC(parvez and(Dr SM. Jl. Jl6idi,for tHeir interest in tfie progress of my research wor^

'thanfis are aCso due to (Dr. %fi.awaja Jamaf, (Dr. Quazi M. JiR, 'Dr. 9\%imat Jiti, (Dr.J^yesfta Qamarwfto afways ftappify e:>(tendedtlieirliefp, u^denever sought for.

I am aCso indeSted to ad my colleagues, juniors and seniors, especiaCfy (Dr. SftaSana Tirdaus, !Ms. (Rana Samad, (Dr. % Vsmani, Mr. CM. J4fzaf "Kfian, !Ms. ShaSihuf (patma JiSidi, JHS. Ufinajilam., !Ms. I'aCat 'Yasmin, for tfieirfriendCy support and cooperation.

'the unstinted cooperation and solicited assistance rendered 6y my friend CHr (Rehan Jafri, and his parents are acHnawCedged with gratitude.

My gratitude linows no Sound when I than({^for the cooperation help and moral support extended to me By my friends, Messers. !Minhaj uddin !MaRf{^ S.^.ldian, JA. Sadat, J^qeeC, Irfan, guCshan (Rjiina, Qaisar S. Mir, M. 'EqSaCSiddiqui, TaisaCJiftmed, ShaSir fi. (Dar, M.(Riaz Kjian and JAsifManzoor "Khan.

'The services rendered 6y Mr 0\''asiruddin, laSoratory assistant is gratefulfy aclinowledged.

In personal note, I express my profound gratitude and gratefulness to my Beloved parents, Brothers and sisters vt^hose Blessings and Boundless affection and encouragement made me to acheive this tas^

Imtiaz yihmed

TO MY (pji'mm's

GENERAL INTRODUCTION

INTRODUCTION

The importance of fish as food has been recognized by man from antiquity. The flesh

of the fish is rich in proteins and minerals like calcium, phosphorous and iron.

Vaiying quantities of fat and oils are also found in fishes. Fish also provide many

other important by-products like fish oil, fish meal, fish flour, fish protein

concentiates, fish glue, fish skin etc. At the time, there has been a continuous increase,

world wide, in the demand for both finfish and shellfish. This has placed an ever-

increasing pressure on producer countries to increase their supply. This rapidly

escalating fishing pressures especially for high value items such as shrimp would

naturally lead to severe over-exploitation beyond the maximum sustainable yield

leading to virtual stock failures in the sea. Therefore, the only solution for these

problems is aquaculture which can give suiprising boost to fish production.

Aquaculture has emerged as one of the most promising industries in the world

for growing fish for the last two decades. The sector is fast approaching a series of

crossroads in its development, where some major decisions will have to be made if it

is to proceed along a suitable developmental path and emerge as one of the world's

major food production systems in the new millennium. The ever increasing demands

of the world's major agricultural food production systems upon a finite quantity of

natural resources (i.e. water, nutiient energy, land) necessitate that farming systems

become increasingly more efficient in term of resources use and have little or no

adverse impact upon society and the environment. Out of 20,000 different species of

the fish only 100 species are cultured for commercial purpose (Sheperd and Bromage,

1988).

Indian freshwater aquaculture has grown rapidly during the last two decades.

The rapid growth of aquaculture has now become the major source of quality protein

for human consumption and a significance source of foreign cunency to many

developing countiies in the international tiade. With availability of the 2-4% of the

global land area, India has to nourish and sustain 16% of the world population.

Consequently more then any other country, India may have to depend more and more

on its water for aquatic food in the years to come. Identically, among the Asian

counties, India ranks second in culture and third in capture fisheries. Fortunately, the

aquatic resources of India are vast and diversified. The fish production in our country

has increased by more than 5 times, and the contribution of fisheries to the GDP of

India has also increased 3 times, a growth arguably one of the highest among the food

production sector. Semi-intensive and intensive culture of freshwater fish is gaining

importance in India. As a result, use of supplementary feeds in aquaculture has

become inevitable for the success of fish culture. The paucity of knowledge on

nutritional requirements, scientific foiTnulations of feeds and data on on-faim feed

evaluation have come on the way of developing cost-effective feed formulations for

larval, giow-out and brood stock fishes which necessitates immediate detailed studies.

Availability of commercial fish feed fonnulations for supplementary feeding that

caters to the nutiient needs of cultured fishes is one of the limiting factors for both

intensive and semi-intensive fish fanning in the country (Devaraj and Seenappa,

1991). Fish raised in a relatively small confinement at high stocking density

(intensive culture) must receive the required nutrients and energy in the form of

protein and amino acids, fat and fatty acids, and carbohydrate. In addition non-energy

nutrients, minerals and vitamins are also adequately required to support life and

promote growth (Halver, 1976).

In culturing fish in captivity, nothing is more important than sound nutiition

and adequate feeding because of their influence on fish growth and health. If the feed

is not consumed by fish or if the fish are unable to utilize the feed because of some

nutrient deficiency, then there will be no growth. An under nourished fish cannot

maintain its health and be productive, regardless of the quality of the environment.

Feeds cost generally constitute the largest variable cost in intensive fish production;

therefore fonnulafion of cost effective feeds can significantly influence the

profitability. One of the challenges is to develop less wasteful, cost effective diets.

Development of nutritious and cost-effective diet is dependent on knowing a species

nutritional requirement and meeting those requirements with balanced feed

formulations and appropriate feeding practices. Although there is an increasing

evidence that gross nutrient requirement of cultured species group are closer to each

other, but the requirements may vary with other factors like fish size, age,

temperature, and nutiient balanced in diet etc.

Formulation of cost-effective diets required accurate information on the amino

acids composition and digestibility of economical ingredients as well as accurate

information on amino acids requirements. Faulty nutiition obviously impairs fish

productivity and results in a deterioration of health until recognizable disease ensues.

The borderlines between reduced growth and diminished health on the one hand, and

overt disease, on the other hand, are very difficult to define. There is no doubt that as

our knowledge advances, the nature of the depaitures from nonnality will be more

easily explained and corrected. However, the problem of recognizing a deterioration

of performance in its initial stage and taking coirective action will remain an essential

part of the skill of the fish cultmist. Lall (2000) mentioned that nutritional status is

considering one of the important factors that deteimined the ability of fish to resist

diseases. Out break of fish diseases commonly occur when fish are stressed due to a

variety of factors including poor nutiition.

Proteins are complex organic compounds of higher molecular weight

containing carbon, hydrogen, oxygen, nitiogen, phosphorous, sulphur and are fonned

of polymerised amino acids. They perfoim essential structural roles and as enzyme are

vital for metabolism. Protein is the major component of most tissues in living

organisms in terms of diy weight. Protein is usually considered to be the most

important nutrient in fish feed and the gross dietary protein requirement is directly

influenced by the amino acids composition of the diet (Keembiyehetty and Gatlin,

1992). Fish generally have higher protein requirement then land animals (Lovell,

1989). In general, proteins do not impose metabolic blocks in fish, because these

animals excrete ammonia as their final catabolic product in protein metabolism.

Therefore, utilization of proteins in fish is extiemely efficient and they can afford

high return rates of free amino acids pools to protein synthesis (Cowey and Luquet,

1983). The high level of free amino acids in body fluids of fish result in regularity

mechanisms, which frequently incapacitate the utilization of dietary free amino acids.

All animal and plant cells contain some protein but the amount of protein

present in food varies widely. It is not just the amount of protein that need to be

considered-the quality of the protein is also important and that depends on the amino

acids, that are present. If a protein contains the indispensable amino acids in the

proportion required by fish, it is said to have a high biological value. If it is

comparatively low in one or more of the essential amino acids it is said to have a low

biological value. The amino acid that is in shortest supply in relation to need is termed

as limiting amino acid. Generally, proteins from animal sources have a higher

biological value than proteins from plant sources, but the limiting amino acid varies.

Lysine is the limiting amino acid in wheat protein, tiyptophan in maize protein, and

methionine and cystine in beef protein.

Amino acid is the fundamental structural components of protein popularly

refened to as the building blocks of protein. Following the digestion of food protein

and absorption of amino acids they can either be incoiporated into body proteins of

fish (anabolism) or further broken down (catabolism) for energy. Although over 100

amino acids have been detected from natural sources, only 20 of those are coinnionlv

found in animal tissue. Physical and chemical differences between the amino acids

give rise to proteins with vaiying sti-uctural configuration. It is these differences,

which play a major role in defining the functions of the protein. Protein is required in

diets to provide indispensable amino acids and niti'ogen for synthesis of non-

indispensable amino acids. Besides being building blocks for tissue proteins and

precursors of many biologically important molecules, some amino acids are

precursors of the purines and pyrimidines needs, for the synthesis of the nucleic acid,

the hereditaiy unit that cany infoimation from one generation to the next generation.

Fish like other animals, do not have a tiiie protein requirement but have a

requirement for a well balanced mixture of essential and non essential amino acids

(Wilson, 1985). Protein in fish body tissue incoiporate about 20 amino acids and

among these 10 amino acids must be supplied in the diet since fish can not synthesise

them in sufficient quantity by the body so that a dietaiy source is required. Dietaiy

requirement for these indispensable amino acids including arginine, histidine.

isoleucine leucine, lysine, methionine, phenylalanine, threonine, tiyptophan. and

valine have been detennined for some fish species. Ten other amino acids that

commonly make up protein can be adequately synthesized by the body and thus are

called dispensable amino acids. These amino acids have nutritional significance

because their presence in the diet consei"ves energy that would be required for

synthesis, and some dispensable amino acids can partially replace or spare

indispensable amino acids, for example methionine and phenylalanine are required as

specific precursors for the synthesis of the dispensable amino acids cystine and

tyrosine, respectively. Teleost fish are known to require the same ten indispensable

amino acids (lAA) as most tenestiial animals, except for arginine, which is essential

in fish much as in tenestiial carnivores (Kaushik, 1998).

Determining a fish's minimum dietaiy requirement for protein or a balanced

mixture of amino acids is critical. Satisfying this requirement is necessaiy to ensure

adequate growth and health of the fish while providing excessive level is generally

uneconomical. Since protein is the most expensive dietaiy component, therefore,

deteiTTiination of the minimum dietaiy protein requirement for a species is generally a

primaiy consideration.

Determination of a specific individual amino acids requirement provides even

more specific infoiTnation about its protein needs. Amino acid requirement constitute

an equally important aspect in nutiition and feed fonnulation of species. Diet deficient

in any one of the 10 indispensable amino acids results in depression of appetite and

reduced weight gain. Replacement of the amino acid results in the recovery of appetite

and growth (NRC 1983). The quantitative amino acid requirement is the minimum

amount of amino acid that gives the maximum growth rate. An excess supply of

essential amino acid over optimum required by fish may not necessarily increase its

utilization (Halver et al, 1957). Detennining the essential amino acid requirements of

cultured fish is of extreme importance due to the significant effects of these nutiients

on muscle deposition, feed cost and nitrogen pollution (Small and Soares, 1999). A

proper balance and quality of essential amino acids may reduce the requirement of

non-essential nitiogen component, thereby, reducing the over all nitiogen requirement

in fish. A deficiency in one or more amino acids may cause poor growth and lack of

appetite (Mertz, 1972).

Development of first successful amino acid test diet for chinook salmon by

Halver et al., (1957) enabled a number of workers to study the essentiality of various

amino acids in other fish species (Halver and Shanks, 1960; Shanks et al, 1962; Nose

et al, 1974; Mazid et al., 1978; Dupree and Halver, 1970 and Ai-ai et al., 1972).

Studies on the quantitative amino acid requirement were mostly based on the test diet

in which the nitiogen component consisted either of all crystalline amino acids or a

mixture of amino acids with selected whole protein source, commonly casein and

gelatin (DeLong el al., 1962, Chance et al., 1964 and Halver, 1965), zein

(Dabrowaski, 1981) or ghiten (Halver et al., 1958, Ketola, 1983). The amino acid

profile of the total protein component of the diet being controlled so as to simulate the

amino acid pattern of the specific reference protein. To assure the maximum

utilization of the amino acids such diet contains protein at slightly below the optimum

requirement.

Measurement of essential amino acid requirement has largely been based on

dose-response cui-ve obtained by feeding graded levels of each amino acid in the test

diet. On the basis of observed growth response plotted over this cui-ve, dietaiy

requirement is usually taken at the breakpoint. Dose-response experiments with

increasing supplies of amino acids are accepted in principle as a method for

detennining requirement (Cowey, 1995).

The other methods such as free amino acid levels within specific tissue pools,

whole blood, plasma or muscle (Kaushik, 1979) or oxidization of radioactive labeled

amino acids administered orally or by injection (Walton et al., 1982) were used as

criteria for estimating the dietaiy amino acid requirements of fish. Using diet

containing a whole protein source with high biological value, Ogino (1980)

deteimined the quantitative essential amino acid requirements of fish on the basis of

the daily deposition of individual amino acids within its carcass. Tacon and Cowey

(1985) refened to a similarity between the relative proportions of individual amino

acids required in the diet and that of the same ten essential amino acids present within

the fish carcass. Wilson and Poe (1985) obtained a sti ong regression coefficient when

essential amino acid requirement of the channel catfish was regressed against the

whole body essential amino acid profile.

Although quantitative essential amino acids requirement have been worked out

for many fish species (Halver cl a/., 1958; 1959; Chance et at., 1964; Delong et al,

1962; Halver, 1965; Meitz, 1969; Dupree and Halver,1970; Klein and Halver, 1970;

Nose, 1970; Cowey and Surgent,1972; Luquet and Sabaut, 1974; Rumsey, 1975;

Harding et al.A911: Wilson et al., 1977; Mazid et al., 1978; Kaushik, 1979; Robinson

et al., 1980"''; Wilson et a/.,1980; Dabrowski, 1981; Murai et al, 1981; Robinson et

al, 1981;Jackson and Capper, 1982; Ketola, 1982; Murai et al, 1982''''; Walton et al,

1982; Hughes, 1983; Ketola, 1983;Kim et fl/.,1983; .launcey et al, 1983; Poston and

Rumsey, 1983; Rumsey el a/., 1983; Kim, et al, 1984; Murai et al, 1984; Robinson et

al, 1984; Walton et al, 1984"^ Walton, 1985; Akiyama et al, 1985"^ Cho and

Woodward, 1985; Thebault, et al, 1985; Murai et al, 1986;Walton et at., 1986;

Hidalgo et al, 1987; Kim, et al, 1987; Murai et al, 1987; Chiu, et al, 1988;

Kaushik and Fauconneau, 1988; Schwarz and Kirchgessner, 1988; Murai et al, 1989;

Viola and Arieli, 1989; Borlongan and Benitez, 1990; D' Mello et al,\990:

Borlongan, 1991; Choo et al, 1991; Moon and Gatlin, 1991; Mohanty and Kaushik,

1991; Tibaldi and Lanari, 1991;Viola and Lahav, 1991; Borlongan, 1992; Cowey et

al, 1992; Cho et al, 1992; Craig and Gatlin, 1992; Griffen et al, 1992

Keembiyehetty and Gatlin, 1992; Kim, et fl/.,1992'^ Pfeffer et al, 1992; Viola et al.,

1992; Anderson et al, 1993; Keembiyehetty and Gatlin, 1993; Khan and Jafri, 1993;

Kim, 1993; Wu, 1993; Cowey, 1994; Griffm el al, 1994'''; Mambrini and Kaushik,

1994; Lall el al, 1994; Lopez- Alvardo and Kanazawa, 1994; Tibaldi et al, 1994;

Boren and Gatlin, 1995; Mambrini and Kaushik, 1995; Murthy and Varghese, 1995;

Rodehutscord el al, 1995; Cai and Burtle, 1996; Mmthy and Varghese, 1996''';

Berge et al, 1997; Davies and Monis, 1997; Keembiyehetty and Gatlin, 1997; Kim,

1997; Murthy and Varghese, 1997'''; Rodehutscord et al, 1997; Ruchimat et al,

1997'^ Twibell and Brown, 1997; Akiyama et al, 1998; Fagbenro et a/., 1998''';

Forster and Ogata, 1998; Kaushik, 1998; Li and Robinson, 1998; Luzzana et al.

1998; Masumoto et al, 1998; Murthy and Vai-ghese, 1998; Schwarz et al, 1998;

Berge, 1999; Coloso et al, 1999; Fagbenro and Nwana, 1999; Fagbenro et al, 1999;

Ngamsnae et al, 1999; Sinmons et al, 1999; Small and Soars, 1999; Tibaldi and

Tulli, 1999; Barziza et al, 2000; Buentello and Gadui, 2000; Kasper et al, 2000:

Rodehutscord, 2000; Rodehutsord et al, 2000; Small and Soars, 2000; Twibell et al,

2000 Yamamoto et al, 2000'''; Alam et al, 2001; GuiTea et al, 2001; and Alam ei

al, 2002. The complete ten essential amino acid requirement have been established

for only a limited number of cuhured fish species including chinook salmon,

Oncorhynchus tshawystscha (Haiver el al, 1957; NRC, 1993), sockeye salmon,

Oncorhynchus nerka (Haiver and Shanks, 1960), coho salmon, Oncorhynchus kisutch

(Arai and Ogata, 1993), common caip, Cypiinus carpio (Nose, 1979), Japanese eel,

Anguilla japonica (Arai et al, 1972; NRC, 1993), rainbow ti-out, Oncorhynchus

mykiss (Ogino, 1980), channel catfish, Ictahmis punctatus (Wilson et al, 1978;

NRC, 1993), Nile tilapia, Oreochromis niloticus (Santiago and Lovell, 1988), Indian

major carp, catla, Catla catla (Ravi and Devaraj, 1991), chum salmon, Oncorhynchus

Keta (Akyama and Arai, 1993), milkfish, Chanos chanos (Borlongan and Coloso,

1993), white sturgeon, Acipenser transmontanus (Ng and Hung, 1995), striped bass,

Morone saxatilis (Small and Soares, 1998).

There is no infoiTtiation available on the amino acids requirement of

commercially important Indian major caip, miigal, Cinhimis mrigala. The present

study was, therefore, undertaken with a view to generate data on its dietary amino

acids requirement and the findings are presented in the form of this dissertation.

The study provides infonnation on the threonine and methionine requirement

oi Cirrhinus mrigala which could be used in developing amino acid balanced diets for

this species.

GENERAL METHODOLOGY

GENERAL METHODOLOGY

Source of fish stock and acclimation.

Induced bred fingeiiings of Indian major caip, Cirrhinus mrigala were procured from

the Hatcheiy of the Uttar Pradesh State Fisheries Department. These were tiansported

to the wet laboratoiy in oxygen filled polythene bags, given prophylatic dip in KMn04

solution (1:3000), and stocked in indoor circular aluminium plastic lining

(Plasticrafts coipn, Mumbai, India, 4ft x 3ft x 3ft) fish tank (water volume 600 L) for

a fortnight. During this period, the fish were fed to satiation a mixture of soybean,

mustard oil cake, rice bran, and wheat bran in the form of moist cake twice a day at

0800 and 1600 hours. These were then acclimated for two weeks on casein-gelatin

based (40% CP) H-440 diet (Halver, 1989) near to satiation.

Preparation of experimental diets

For studying the essential amino acids requirement of Cirrhinus mrigala, test diets

containing graded levels of the amino acids under study were used. The dietary range

necessary to quantify the requirement of amino acids was adjusted on the basis of

infomiation available on other fish species.

Calculated quantities of ciystalline amino acids (Chance et al, 1964) and salt

mixture (Halver, 1989) were thoroughly stined in a volume of hot water (80 °C) in a

steel bowl attached to a Haboit electiic mixer. The pH of the resulting mixture was

adjusted to neutial with 6N NaOH solution (Nose et al, 1974). Gelatin powder was

dissolved separately in a volume of water with constant heating and stirring and then

tiansfeiTed to the above mixture. Other diy ingredients and oil premix, except

carboxymethyl cellulose, were added to the lukewarm bowl one by one with constant

mixing at 40°C temperature. Carboxymethyl cellulose was added in the last and the

speed of the blender was gradually increased as the diet started to harden. The final

diet, with the consistency of bread dough, was poured into a teflon-coated pan and

placed into refrigerator to gel. The prepared diet was in the foi-m of moist cake (50%

diy matter) from which cubes were cut and stored at -20 °C in sealed ploythene bags

until used.

Feeding trial

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

maintained in the wet laboratory. These were stocked in tiiplicate groups in 70 L high-

density polyvinyl circular houghs (water volume 55 L) fitted with a continuous water

flow through system. The water exchange rate in each trough was maintained at 1.0-

1.5 L/min. The feeding schedule and levels were chosen after carefully observing the

feeding behaviour of the fish and their intake. As per the result obtained in the

preliminary feeding trial, fish were fed test diet in the form of moist cake at the rate of

5% of the body weight six day a week twice a day at 0800 and 1600 h. The feeding

trials lasted for six weeks. Initial and weekly fish weights were recorded on the top

loading balance (Precisa 120 A). Troughs were siphoned with a pipe to remove fecal

matter or other things, if any, before feeding daily. Accumulation of the diet at the

bottom was avoided. Any uneaten food was siphoned immediately over a pre-weighed

filter screen, 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

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

the assessment of carcass body composition.

Physico-chemical condition of the rearing water.

Dissolved oxygen was measured using Winkler's titrimetric methods (APHA, 1992)

daily in the morning.

No. of ml of Na2S203 solution consumed x 1000 x 8 Dissolved oxygen = mg/1

50x40

During the period of experiment, water temperature was recorded daily at 7 AM, 12

Noon and 12 PM using a mercury themiometer of 0.1 "C accuracy.

pH of the water was measured by a digital pH meter (Eutcheberentics Pte Ltd.,

Singapore) with an accuracy up to 0.1.

The dissolved free carbon dioxide was measured using phenolphthalein indicator and

titration (APHA, 1992) daily in the morning at 7AM and expressed as mg/1.

No. of ml of N/44 NaOH required x 1000 x 44 D. Free Carbon di-oxide = mg/1

50x44

Total alkalinity was measured using phenolphthalein indicator.

No. of ml of N/50 H2SO4 consumed xlOOO Total alkalinity = mg/1

ml of sample

Proximate analysis

Assessment of proximate composition of feed ingredients, experimental diets, fish

carcass was made using the standards techniques (AOAC, 1995). All chemical

analysis were based on tiiplicate sample.

Moisture

Known quantity of sample was taken in a pre-weighed silica cmcible and placed in

hot air oven at 105±l"C for 24 hours. The crucible containing the dried sample was

cooled in desiccator and reweighed to ensure that the sample had become completely

dried. The loss in weight gives an index of water from which its percentage was

calculated.

12

Ash

Dried powdered sample (2-5g) was taken in a pre-weighed silica crucible and

incinerated in a muffle furnace (600 °C ) for 2-4 hours or till the sample became

completely white and free of carbon. The crucible was cooled in a desiccator and

reweighed to estimate the quantity of ash. The result was expressed as per cent on diy

weight basis.

Fat

Cmde fat was estimated by continuous soxhlet extiaction technique using petioleum

ether (40-60 °C B P) as solvent. Finely powdered and dried sample (2-4g) was placed

in Whatman fat extiaction thimble, plugged with cotton, and placed in the soxhlet

apparatus. A clean, diy soxhlet receiver flask was weighed and fitted to the soxhlet

assembly on a boiling water bath for extiaction which was continued for 10-12

hours. After exti'action the flask was removed and kept in a hot air oven (100 °C) to

evaporate the traces of solvent. It was then transfened to a desiccator, cooled and

reweighed. The difference between the weight of the flask before and after the

extiaction gave the quantity of crude fat extracted from the unknown amount of

sample. The result was expressed as per cent on diy weight basis.

Crude proteni

The estimation of crude protein was canied out by slightly modifying the Wong's

microkjeldhal methods as adopted by Jafri et al. (1964). The principle of this

technique involves digesting a known quantity of sample with N-free sulphuric acid in

presence of a catalyst, potassium persulphate which convert the nitrogenous

compound to ammonium sulphate. This is then nesslerized and the colour developed,

due to the fonnation of complex compound (NHgal), is measmed

spectrophtometrically. The optical density is read off against a standard curve of

(NH4)2 SO4 for nitrogen estimation from which the amount of crude protein is

calculated.

13

A known quantity of dried, finely giound sample (0.1- 0.5g ) was taken in a

kjeldhal flask containing 5ml of nitrogen-free sulphmic acid (1:1) and heated till

fumes disappeared. After cooling, 0.5ml of nitrogen-free saturated potassium

persulphate was added to oxidize the digesting mixture. The digestion was continued

for about 12-14 hours or till the solution in the kjeldhal flask became water clear,

indicating that all the nitrogenous material present in the sample has been converted

into ammonium sulphate. After cooling, the digested mixture was transferred to a 50

ml volumetric flask and raised to mark with double distilled water. 0.5 ml of aliquot of

the digested sample was taken in a diy test tube and 0.1 ml each of sulphuric acid

(1:1) and saturated potassium persulphate solution added to it. The volume was raised

to 3 ml with double distilled water. This was than nesselerized with 7 ml of Bock and

Benedict's Nessler's reagent (Oser, 1971). The solution was kept at room temperature

for 10 minutes for complete colour development. A blank was prepared side by side

substituting the aliquot with distilled water. The absorbance was measured after

setting the instrument with a blank at 480 nm. A microprocessor controlled split beam

spectronic 1001 spectiophotometer (Milton Roy Company, USA) was used. The

intensity of colour developed was proportional to the amount of ammonium sulphate

contained in the solution. The value of optical density obtained for various samples

were read off against a standard calibration cui ve (Fig.l) prepared by taking readings

of a series of different dilution containing different grades of known amount of

nitrogen which was multiplied with conventional protein factor (6.25X N) to obtain

the cmde protein content in the sample. The values were recorded as percentage on

dry weight basis.

Estimation of energy

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

Loughborough, England). Prior to estimate, a weighed quantity of dried powdered

sample (0.5-l.Og) was taken in a metallic crucible and compacted carefully to increase

the rate of combustion at 25 lb oxygen pressure.

14

The heat generated upon combustion was read on the modulated galvanometer

scale, and converted to energy equivalent, workout 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 the test diet were calculated as 4.83, 5.52, 5.8,

3.83,and 9 kcal/g for gelatin, casein, amino acid, dextrin and fat respectively, as

estimated on Gallenkamp ballistic bomb calorimeter.

Assessment of growth and conversion efficiencies

Calculation of various growth parameters, conversion efficiency, nutrient retention,

and biological indices were made according to the following standard definitions

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

Weight gain (%)

=100 X (final Body weight-initial Body weight)/initial Body weight.

Specific growth rate (%)

= 100 X (In final wet weight (g)-ln initial wet weight g)/duiation (days)

Feed conversion ratio (FCR)

=Diy weight of feed consumed / Wet weight gain

Protein efficiency ratio (PER)

=Wet weight gain (g) / Protein consumed (g, dry weight basis)

Protein deposition

=100 X ( BWf X BCPf) - ( BWi X BCP;) / [TF X CP]

Where BW; and BWf = mean initial and final body weight (g), BCP, and BCFf = mean

initial and final percentage of muscle protein, TF =Total amount of diet consumed,

and CP=Percentage of cmde protein of the diet.

15

Statistical analysis

The data was statistically analysed using central computer facility of the university.

Responses of fmgerling CArrhinus mrigala to graded levels of the test amino acids

were measured by weight gain (%) feed conversion ratio (FCR), protein efficiency

ratio (PER), specific growth rate (SGR) and body composition. These response

variables were subjected to one-way analysis of variance (ANOVA) (Sokal and Rohlf,

1981; Snedecore and Cochran, 1967). To detennine the significant differences among

the tieatments, Duncan's Multiple Range Test (Duncan, 1955) was employed. Broken

line model (Zeitoun et al, 1976) was used to find out the break point in the growth

curve. The break point obtained represented the optimum requirement of the fish for

the amino acid under study. Second-degree polynomial regiession analysis (Y=

a+bx+cx^) was employed to FCR to predict more accurate response to the dietary

intake. Simple regression (Y= a+bx) and conelation coefficient (r) were employed.

For statistical analysis software package Metlab, SPSS and Sigma plot were used.

16

Table 1. Composition of mineral mixture*

Minerals g/lOOg

Calcium biphosphate 13.57

Calcium lactate 32.69

Ferric citi ate 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

Manganous sulphate H2O 0.080

Cobalt chloride 6H2O 0.100

Zinc sulphate 7H2O 0.300

*Haliver(1989)

Table 2. Composition of vitamin mixture''

Vitamins g/lOOg

Alpha cellulose

Choline chloride

Inositol

Ascorbic acid

Niacin

Calcium pantothenate

Riboflavin

Menadione

Pyridoxine HCI

Thiamin HCI

Folic acid

Biotin

a-Tocopherol acetate

Vitamin B12***

* *

*Halver, 1989. **Incorporated with oil. ***(10mg/500mlH2O).

8.000

0.500

0.200

0.100

0.075

0.050

0.020

0.004

0.005

0.005

0.0015

0.0005

0.040

0.00001 (0.5 ml)

Table 3. Composition of the amino acid mixture*

Amino acids

(L-series)

Arginine

Histidine

Isoleucine

Leucine

Lysine monohydrochloride

Methionine

Cystine

Phenylalanine

Tyrosine

Threonine

Tryptophan

Valine

Alanine

Aspartic acid

Glutamic acid

Proline

Serine

Glycine

Total

Amino acids

(g/100 g)

8.299

2.371

7.588

8.726

9.829

3.913

2.276

5.976

4.268

4.102

1.422

6.924

5.454

3.272

6.711

7.968

1.636

12.259

100.000

Protein supplied by amino

acids (g/100 g)

16.680

4.013

5.064

5.823

6.546

2.295

1.658

3.167

2.062

3.015

1.219

5.174

5.359

2.152

3.993

6.058

1.363

14.295

89.936

* Calculation based on Chance et ai, 1964.

90 100 110

Fig. 1 Calibration curve of nitrogen.

Flow-through system used for feed trials

Fish being acclimatized on experimental diet

o n'

C/5

o o 5«r CD

Experimental diet and initial pooled carcass samples

Final pooled carcass samples

CHAPTER 1

CHAPTER 1

DIETARY THREONINE REQUIREMENT OF FINGERLING INDIAN

MAJOR CARP, CIRRHINUS MRIGALA (HAMILTON)

INTRODUCTION

Precise information on nutritional requirements of culturable fish species is essential

to reduce feed cost by allowing provision of appropriate amounts of various nutrients

for optimal growth and health. Of the nutiients required by fish, the indispensable

amino acids, that make up quality protein an expensive component in fish diet, aie of

utmost importance because of their influence on fish growth and diet economics

(NRG, 1993).

In inland fisheries sector, Indian major caips, namely, catla, Catla catla, rohu,

Labeo rohita and miigal, Cirrhinus mrigala, comprise the major capture / culture

fisheries (Jhingran, 1991). These caips are fast growing attaining marketable size of

800-1000 g in less than a year and are generally propagated on extensive and/ or

intensive scales in a polyculture system (Jhingran and Pullin, 1988). Previous studies

on the nutrition of these fishes remained confined to their protein and amino acid

requirements (Sen et al, 1978; Renukaradhy and Varghese, 1986; Singh et al, 1987;

Singh and Bhanot, 1988; Shetty and Nandeesha, 1988; Swamy et a/., 1988,1989;

Mohanty et al, 1990; Das and Ray, 1991; DeSilva and Gunasekera, 1991; Khan and

Jafri, 1991; and Jena et al., 1996). Cirrhinus mrigala is used as a component of

polyculture with other species of major caips.

Quantitative dietary amino acid requirements for ten amino acids have been

investigated for several aquaculture species and reviewed under General Introduction

section (page 8).

After lysine and methionine, threonine is the most limiting indispensable amino

acid in practical ingredients used for diets preparation. Dietary threonine

requirements have been worked out for Chinook salmon, (Halver et al, 1958; DeLong

et al., 1962), Mossambique tilapia, Oreochromis mossambicus (Jauncey, 1983), chum

salmon, (Akiyama et al, 1985"), Indian major carp, catla, (Ravi and Devaraj,1991),

red diiim, Sciaenops ocellatus (Boren and Gatlin, 1995), Indian major carp, rohu,

Laheo rohita (Murthy and Varghese, 1996), juvenile stiiped bass, Morone chrysops x

M.saxatilis (Keembiyehetty and Gatlin, 1997), stiiped bass, Morone saxatilis (Small

and Soais, 1999), European stiiped bass, Dicentrarchus labrax (Tibaldi and Tulli,

1999).

Although protein requirement of the Indian major carp, Cirrhinus mrigala has

been quantified (Swamy et al, 1988; Mohanty et al, 1990 ; Das and Ray, 1991 and

Khan and Jafri, 1991), no information is available on its essential amino acid

requirement. Hence, the present investigation was undertaken to determine the

optimum dietaiy threonine requirement of fingerling Cirrhinus mrigala.

MATERIALS AND METHODS

Experimental diet

Six isonitrogenous (40% CP) and almost isocaloric diets (4.28 kcal g"\ GE) with

graded levels of threonine were formulated, using casein (fat free), gelatin and L-

crystalline amino acid premix (Table I). The dietary protein level was fixed at 40%,

reported optimum for the growth of Cirrhinus mrigala (Khan and Jafri, 1991). L-

crystalline amino acids were used to simulate the amino acid profile of the diets to that

of 40%) whole chicken egg protein, excluding the test amino acid (threonine). The

levels of L-threonine were in increment of 0.25g/100g of diy diets. A casein-gelatin

ratio, contiibuting minimum quantity of the test amino acid and maximum quantities

of other amino acids was maintained. The quantity of threonine was increased at the

expense of glycine so as to make the diets isonitrogenous. The levels of threonine in

the test diets were fixed on the basis of information available on the other Indian

major carps, Catla catla (Ravi and Devaraj, 1991) and Labeo rohita (Murthy and

Varghese, 1996).

Method of preparation of the experimental diets has been described under

General Methodology section (page 10-11). Proximate composition of the diets and

body carcass were estimated according to standard methods (page 12-15).

Feeding trial

Source offish, their acclimation, and details of general experimental design have been

described under General Methodology Section (page 10-11).

Cirrhinus mrigala fmgerling (4.35+ 0.80 cm, 1.08 ± 0.30 g) were then stocked

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

a continuous water flow through (1-1.5 1/min) system at the rate of 15 jfish per trough

for each dietary treatment level. Fish were fed test diets in the form of moist cake at a

rate of 5% body weight, twice daily at 0800 and 1600 h. No feed was offered to the

fish on the day the measurements were taken. Initial and weekly weights were

recorded on a top loading balance (Precisa. 120A) and feed allowances adjusted

accordingly. The feeding tiial lasted for six weeks. Faecal matter and unconsumed

feed, if any, were siphoned before feeding. Water temperature, pH, free carbon

dioxide, dissolved oxygen and total alkalinity during the feeding trial were recorded

following the standard methods (APHA, 1992). The average water temperature,

dissolved oxygen, free carbon dioxide, pH, alkalinity over the six weeks feeding trial,

based on daily measurements, were 26-27.5 °C, 6.62-7.4 ppm, 06-12 mg/1, 7.2-7.8 and

46-65 mg/1, respectively.

Growth parameters and feed utilization efficiencies were measured using

standard definitions have been given elsewhere (page 15).

Chemical analysis

Proximate composition of casein, gelatin, experimental diets, initial, final carcass

and gross energy was estimated using standai'd AOAC (1995) methods (page 12-15).

Before the commencement of the feeding trial, desired number of fish were randomly

sacrificed and pooled sample in triplicate were taken for the determination of initial

whole body composition. At the end of the feeding trial fish from each dietary

treatment were randomly taken out and analyzed for their final carcass composition

and gross energy. Whole body energy were determined in Gllenkamp blastic bomb

calorimeter (page 14-15). Amino acid analysis of casein and gelatin was made with

the help of Beckman System Gold HPLC unit.


The data were statistically analyzed using one-way analysis of variance (ANOVA),

followed by Duncan's Multiple Range Test at 0.05% probability level (Snedecor and

Cochran, 1967; Sokal and Rohlf, 1981; and Duncan, 1955). Break point regression

analysis (Zeitoun et al, 1976) and second-degree polynomial regression analysis were

also employed.

RESULTS

Growth performance of fingerling Cirrhinns mrigala fed diets containing graded

levels of threonine for six weeks are shown in Table 2. Maximum weight gain (%),

specific growth rate (SGR%), protein efficiency ratio (PER), and best food conversion

ratio (FCR) were obsei-ved in fish fed diet containing 1.75% thieonine (diet IV). Poor

growth and efficiency of feed utilization were evident below and beyond this level of

threonine inclusion. No mortality was obsei-ved in fish fed the experimental diets

during the length of the experiment. On subjecting the growth data to broken line

analysis (Zeitoun et al., 1976), a break point was evident at 1.70% dietary threonine,

corresponding to 4.25% of dietary protein (Fig. 1).

20

The FCR in Cirrhinus mrigala fingerling fed 1.75% threonine diet differed

significantly (P<0.05) from the other levels of threonine inclusion, except 1.50%

(Table 2). The FCR (Y) to dietary levels of threonine (X) relationship was best

described by a second order polynomial curve (Fig. 2) and the following mathematical

relationship

Y= 4.2257X^-14.3141X +3.6816 (r =-0.3483)

Based on the above equation, the best estimated FCR occurred at threonine

level of approximately 1.70% of diet (Fig. 2). The best PER and SGR (%) were also

found in fish fed 1.75% thieonine diet, and these were significantly higher (P<0.05)

than the values obtained with other diets (Table 2).

Carcass composition of Cirrhinus mrigala fed diets containing different levels

of threonine are presented in Table 3. Carcass protein deposition was found to be

significantly higher (P<0.05) at 1.75% dietaiy thieonine (Fig. 3). The carcass moisture

decreased with increasing dietaiy thieonine levels up to 1.75% and thereafter an

insignificant (P>0.05) increase was evident. No significant difference in carcass fat of

fish fed diets containing different dietary thieonine levels were noticeable except at

1.00% threonine inclusion where a significantly (P<0.05) lower fat value was seen.

Fish receiving different thieonine levels, with the exception of those fed 1.00 and

1.75% diets, showed no significant differences in their ash content. However, 1.00

and 1.75% dietary threonine levels produced significantly (P<0.05) highest and lowest

ash values in fish, respectively.

DISCUSSION

Growth is the result of intake of the indispensable amino acids resulting from high

protein deposition. Deficiency of indispensable amino acids may cause reduced

growth, poor feed conversion, and some deficiency symptoms (Wilson and Halver,

21

1986). The utilization of amino acids depends, in general, upon the level of their

intake and balance in diet.

Inclusion of optimum amount of threonine is prerequisite to the formulation of

nutritionally adequate artificial feeds for the culture of a fish species. The present

finding indicates that 1.75% dietaiy threonine is optimum for maximum growth of

fingerling Cirrhinus mrigala. Best feed efficiency, expressed as feed conversion ratio,

is the reflection of highest weight gain, best specific growth rate and protein efficiency

ratio at the above level of dietaiy threonine. However, broken-line regression analysis

of the growth-response cui-ve at 1.70% dietary threonine (Fig.l), is taken as the

optimum dietaiy requirement for this species. The break point obtained at 1.70%

threonine inclusion in the diet indicates that increasing threonine beyond this level

could not produce significant additional growth. This value, corresponding to 4.25%

of dietaiy protein, is close to the requirements reported for the Indian major carp, rohu

4.28% (Murthy and Varghese, 1996); common carp 4.30% (Nose, 1979), but is

higher to the requirements reported for chinook salmon 2.25% (De Long et al., 1962);

channel catfish 2.20% (Wilson et al, 1978); Japanese eel 3.90% (Nose, 1979);

rainbow trout 3.40% (Ogino et al, 1980); Mossambique tilapia 2.23% (Jauncey et al,

1983); chum salmon 3.00% (Akiyama et al, 1985); Nile tilapia 3.75% (Santiago and

Lovell, 1988); red dmm juveniles 2.28% (Boren and Gatlin III, 1995); juvenile striped

bass 2.60% (Keembiyehetty and Gatlin, 1997); juvenile European sea bass 2.60%

(Tibaldi & Tulli 1999); striped bass 2.45% (Small and Soares, 1999). The threonine

requirement obtained for Cirrhinus mrigala is, however, lower than the requirements

reported for Indian major carp, catla 4.95%) (Ravi and Devaraj, 1991) and milkfish

4.50% (Bortongan and Coloso, 1993).

Carps do not have an acidic stomach and digestion proceeds in a neutral or

slightly alkaline environment. The consumption of an acidic amino acid diet with a

strong buffering capacity might disturb the noimal assimilation of the ingested amino

acids. Murai et al, (1981) suggested that carp fingerling fed gelatin diet.

22

supplemented with essential amino acids coated with casein, and casein-gelatin

control diet produced a faster growth than fish fed all ciystalline amino acids in the

diet, and also suggested that amino acid coating by casein alters the relative absorption

rate of certain amino acids in the gut. Additional evidence in support of this

hypothesis was obtained by Murai et al., (1982) who compared the change in the

plasma free amino acids after feeding diets containing casein coated and uncoated

amino acids. It was suggested that the poor utilization of crystalline amino acids by

caips could be the result of rapid excretion which reduced the utilization of these

amino acids for growth (Murai el al, 1984). Khan and Jafri (1993) pointed out that in

comparison to all crystalline amino acid diet, casein-gelatin coated diet indicated

improved utilization of crystalline amino acids in Labeo rohita, presumably through

increased retention in the gut. Hence, in the present study, dietary threonine

requirement of Cirrhinus mrigala was determined using neutralized (6N NaoH)

casein-gelatin based amino acid test diet.

The wide variations obsei'ved in the requirement levels for threonine among

different fish species may be due to the differences in size, age, laboratory conditions,

including feeding regime, feed allowance, water temperature, flow rate, stock density,

as also to the use of various ingredients for basal diets such as casein, gelathi, zein,

gluten, fish meal, soybean meal and crystalline amino acids in varying combinations

as protein sources. Formulation of amino acid test diets from purified and practical

ingredients deficient in test amino acid differs markedly with respect to their nutrient

content, digestibility and amino acid profile and these may also alter the dietary amino

acid requirement values (Simmons et al., 1999; De Silva et al., 2000). Dietary amino

acid requirement is also reported to be influenced by feeding levels adopted (Baker,

1984; Chiu et al., 1988). In the present study on Cirrhinus mrigala feeding regime and

ration size were worked out in a preliminaiy feed trial for ensuring faster feed

consumption and growth. Decreased growth rate in fish fed diets with higher levels of

threonine may be due to toxic effect (Cho et al., 1991) and stress caused by excess

threonine in the body offish, leading to extra energy expenditure towards deamination

23

and excretion of the same (Walton, 1985). Except for poor growth and low feed

efficiency, no pathological symptoms were observed in Cirrhinus mrigala fmgerling

fed threonine deficient diet.

Dietaiy thieonine requirement of fmgerling Indian major carp, Cirrhinus

mrigala was found to be 1.70% of the dry diet, corresponding to 4.25% of dietary

protein. Data generated in the present study on quantitative dietary threonine

requirement of Cirrhinus mrigala would be useful in developing threonine balanced

practical diets for the intensive culture of this species.

SUMMARY

Indian major carp fmgerling, Cirrhinus mrigala (4.35±0.80 cm, 1.08±0.30 g) were fed

isonitrogenous and isocaloric diets (40%) C.P, 428.34 kcal /lOO g, GE) containing

casein, gelatin and ciystalline amino acids with graded levels of L-threonine (1.00,

1.25, 1.50, 1.75, 2.00 and 2.25 g/100 g, dry diet) to determine its dietary threonine

requirement. Feeding tiial was conducted in triplicate for six weeks. Diets were fed

twice a day at 08:00 and 16:00 h at 5% body weight/day. The ration size and feeding

schedule were worked out prior to the start of the feeding trial. Weight gain (148%)

and feed conversion ratio (1.49) were significantly higher (P<0.05) in fish fed diet

containing 1.75%) dietaiy threonine. Broken-line analysis of the weight gain data

indicated the dietary threonine requirement to be 1.70 g /100 g of the dry diet,

corresponding to 4.25%o of dietary protein. Second-degree polynomial regression

analysis was employed to determine the relationship between food conversion ratio

(FCR) and dietaiy threonine levels, which also indicated that the best FCR occuned at

approximately 1.70% dietaiy methionine level. Significant (P<0.05) variations were

also noted in carcass composition of fish fed diet containing graded levels of dietary

threonine. Water temperature, dissolved oxygen, dissolved free carbon dioxide, pH

and total alkalinity were checked regularly during the experiment. No mortality was

observed during the length of the feed trial.

24

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67.9133 + 127.08 X$ 1.70 67.9133 +127.08X>: 1.7 0 390.72 + U 3 . 5 8 X > 1.7 0

1-00 1.25 1.50 1.75 2.00

THREONINE LEVEL (» / ,d ryd ie t )

2.25

Fig. 1.

Fig. 1. Relationship between live-weight gain per cent and dietary threonine levels.

160 -

1 Y =

2 Y =

3 Y =

67.9133 + 127.08 X$ 1.70 67.9133 +127.08X>: 1.70 390.72 + U 3 , 5 8 X > 1.7 0

1-00 .25 1.50 175 2.00

THREONINE LEVEL (»/, dry d ie t )

2.25

Fig. 1.

Fig. 2. Second-order polynomial relationship of food conversion ratio to threonine

levels.

1.2 1.4 1.6 1.8 2

THREONINE LEVEL (% dry diet)

2.2

Fig. 2.

Fig. 3. Relationship between protein deposition and dietaiy threonine levels.

40

30

z O H W O a. I l l

Q 20 L U i

S I ^ I W i

< I o 6 10 ^

1.25 1.5 1.75 2

THREONINE LEVEL (% dry diet)

2.25

Fig. 3.

CHAPTER 2

CHAPTER 2

DIETARY METHIONINE REQUIREMENT OF FINGERLING INDIAN

MAJOR CARP, CIRRHINUS MRIGALA (HAMILTON)

INTRODUCTION

Indian major caips, the prime cultivable freshwater fishes are raised in India on simple

supplementary dietaiy combinations of plant and animal feedstuffs (Jhingran, 1991).

These carps are fast growing attaining marketable size of 800-lOOOg in less than a

year and are generally propagated on extensive and/or intensive scale in a polyculture

system (Jhingran and Pullin, 1988). Intensive cultivation of these fishes is hampered

mainly because of the non-availability of balanced rations formulated on the basis of

their nutritional requirements.

Fish do not seem to have a trne protein requirement but a well balanced

mixture of indispensable amino acids in the diet is essential for their growth and

maintenance. Since protein is the most expensive component of feed items, inclusion

of its optimum amount will enable us to develop cost-effective dietary formulation. To

optimize the protein level in Cirrhinus mrigala diet, precise data on the essential

amino acids requirement are needed. Determining the essential amino acids

requirements of cultured fish is of extieme importance due to the significant effects of

these nutrients on muscle deposition, feed costs and nitrogen pollution (Small and

Soares, 1999). Quantitative dietary amino acid requirements have been investigated

for several cultured fish species (NRC, 1993).

All animals, including fish, are known to require methionine as an

indispensable amino acid in their dietaiy protein (Ketola, 1982; Lovell, 1989). The

essential amino acid methionine is important because it is used for protein synthesis,

converted to cystine for incoiporation into protein, and in the form of S-adenosyl-

metliionine, it is the principal donor of metliyl groups in tlie body. The methyl carbon

of methionine is the source of one carbon group that conjugate with glycine to

produce serine. As S-adenosyl methionine, methionine is the precursor of the

polyamines speimine and speimidine which have diverse physiological role related to

cell proliferation and growth (Munay et al, 1996). Methionine is also a precursor of

choline. Kasper et al. (2000) reported that when methionine is not in excess in the diet

of nile tilapia, choline is required for growth. In general, methionine is the first

limiting amino acid in the ingredients used for fish diet formulation (Alexis et al,

1985; Poston, 1986). Methionine deficiency results in slow growth and reduced feed

efficiency and leads to the development of lanticular cataracts in several sahnonid

species (Walton el al., 1982; Rumsey et al, 1983; Cowey and Tacon,1983;

Keembiyehetty and Gatlin, 1993).

The requirements for methionine differ among various species as well as with

the level of dietary cystine with published value ranging from 2.0 to 4.0% of protein

(Wilson and Halver, 1986). The methionine requirement has been determined for

cultured fish species such as gilthead sea bream, Sparus aurata (Luquet and Sabaut,

1974), rambow trout (Walton et al, 1982; Rumsey et al, 1983; Kim et al, 1992),

mossambique tilapia, Oreochromis mossambicus (Jackson and Capper, 1982; Jauncey

et al, 1983), European sea bass, Dicentrachus labrax (Thebault et al, 1985), juvenile

red drum, Sciaenops ocellatus (Moon and Gatlin, 1991), juvenile hybrid striped bass,

Morone chrysops x M. saxatilis (Keembiyehetty and Gatlin, 1993; NRC, 1993),

juvenile hybrid striped bass, Morone saxatilis x M. chrysops (Griffin et al, 1994^),

channel catfish (Cai and Burtle, 1996), common carp (Schwarz et al, 1998), Indian

major carp, rohu, Labeo rohita (Khan and Jafri, 1993; Murthy and Varghese, 1998),

African catfish, Clarias gariepinus (Fagbenro et al, 1998 "), Asian sea bass, Lates

calcarifer (Coloso et al, 1999), juvenile arctic charr, Salvelinus alpinus (Simmons et

al, 1999), juvenile yellow perch, Perca flavescens (Twibell et al, 2000), and

Japanese flounder, Paralichthys olivaceus (Alam et al, 2001).

26

Cirrhinm mrigala, commonly known as mrigala, is a fast growing fish species,

which is cultured in the Indian sub-continent with other indigenous and exotic species

of carps. Although protein requirement of Indian major carp, Cirrhinus mrigala has

been worked out (Swamy et ai, 1988; Mohanty et al., 1990; Das and Ray, 1991 and

Khan and Jafri, 1991), no infoiination is available on its essential amino acids

requirement. Hence, the present investigation was undertaken to work out the

optimum dietaiy methionine requirement of fmgerling Cirrhinus mrigala.

MATERIALS AND METHODS

Experimental Diet

Six isonitrogenous (40 % CP) and almost isocloric (4.28 kcal g^', GE ) diets with

graded levels of methionine were foimulated using casein (fat-free), gelatin and L-

ciystalline amino acid premix (Table 1). The dietary protein level was fixed at 40%

CP, reported optimum for growth of Cirrhinus mrigala (Khan and Jafri 1991). L-

crystalline amino acids were used to simulate the amino acid profile of the diets to that

of 40% whole chicken egg protein, excluding the test amino acid (Methionine and

cystine). The levels of L-methionine were in increment of 0.25g / 100 g with 1.00 %

cystine fixed in all the diets. A casein-gelatin ratio, contributing minimum quantity of

the test amino acid and maximum quantities of other amino acids was maintained. The

quantity of methionine was increased at the expense of glycine so as to make the diets

isonitrogenous. oc-Cellulose was used to make the diets isoenergetic (Table 1).

The level of methionine in the amino acid test diets were fixed on the basis of

information available on the other Indian major carps, Catla catla (Ravi and Devaraj,

1991) and Labeo rohita (Khan and Jafri, 1993 and Murthy and Varghese, 1998).

Methods for preparation of experimental diets has been described under

General Methodology section (page 10-11). Proximate composition of the body

27

carcass and experimental diet were estimated according to standard methods (page 12-

15).

Feeding Trial

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

given elsewhere (page 10-11).

Cinhimis mrigala fmgerling (3.85± 0.50 cm, 0.50+ 0.02 g) were then stocked

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

a continuous water flow through (1-1.51 / min) system at the rate of 20 fish per trough

for each dietary treatment level. Fish were fed test diets in the form of moist cake at a

rate of 5% of body weight, twice daily at 0800 and 1600 h. No feed was offered to the

fish on the day the measurement was taken. Initial and weekly weights were recorded

on a top loading balance (Precisa. 120A) and feed allowances adjusted accordingly.

The feeding trial lasted for six weeks. Fecal matter and unconsumed feed, if any, were

siphoned before feeding. Water temperature, dissolved oxygen, free carbon dioxide,

pH, total alkalinity during the feeding trial, were recorded following the standard

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

carbon dioxide, pH, total alkalinity over the six weeks feeding, trial based on daily

measurements, were 26.0 -27.5 ' C, 6.7-7.6 ppm, 5-10 mg/1, 7.1-7.8 and 65- 80mg/l,

respectively.

Chemical analysis

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

gross energy were estimated using standard AOAC (1995) methods (page 12-15).

Before the commencement of the feeding trial, desired number of fish were randomly

scarified to and pooled sample in tiiplicate were taken for the determination of initial

whole body composition. At the end of the feeding trial fish from each dietary

tieatment were randomly taken out and analyzed for their final carcass composition

and gross energy. Whole body energy was determined in Gllenkamp blastic bomb

28

calorimeter, (page 14-15) Amino acid analysis of casein and gelatin was made with

the help of Beckman System Gold HPLC unit.


Comparison among different treatment means were made by one-way analysis of

variance (ANOVA) and Duncan's Multiple Range Test (P< 0.05%) was employed to

test the homogeneity of variance with in the treatments. Broken line regression

analysis and second-degree polynomial regression analysis was also employed.

RESULTS

Over the 6 weeks growth trial, significant differences were observed in live weight

gain per cent of fingerling Cirrhinus mrigala fed diets containing different levels of

dietary methionine with 1.00% cystine fixed in test diets (Table 2). Fish receiving

1.00% methionine diet reflected a maximum gain in weight while those fed diets with

higher and lower methionine levels showed reduced weight gain and efficiency of

feed utilization. The highest specific growth rate per cent (SGR %), protein efficiency

ratio (PER), and best feed conversion ratio (FCR) were also obsei-ved in fish fed diet

containing 1.00% methionine (Diet III). No mortality was obsei-ved in fish fed all the

experimental diets. On subjecting the growth data to broken line analysis (Zeitoun et

a/., 1976), a break point was also evident at 1.00% dietary methionine level,

con-esponding to 2.50% of dietaiy protein (Fig. 1).

The feed conversion ratio (FCR) in Cirrhimis mrigala fingerling fed 1.00%

methionine diet differed significantly (P< 0.05) from the other dietaiy levels of

methionine inclusion except 1.25% methionine level diet (Table 2). The FCR (Y) to

dietaiy levels of methionine (X) relationship was best described by a second order

polynomial cui-ve (Fig. 2) and the following mathematical relationship.

Y - 2.781X^ - 6.7215X + 5.562 (r = 0.1694)

29

Based on the above equation, the best estimated FCR occurred at a dietary

methionine level of approximately 1.09 % of diet (Fig. 2). The best PER and SGR %

were also found in fish fed 1.00% methionine diet and these were significantly higher

(P<0.05) than the values obtained with the other diets (Table 2).

Carcass composition (Table 3) of fmgerling Indian major carp, Cirrhinus

mrigala fed varying levels of dietary methionine varied significantly (P<0.05) except

ash content which was insignificantly different among various dietary groups. Protein

content of fish fed diet containing 1.00% methionine was significantly (P<0.05)

higher followed by those receiving diets containing 0.75 and 1.25% methionine level.

However, lower carcass protein was noted in fish fed diet containing 0.5%

methionine, which was insignificantly different with that of 1.50% methionine.

Carcass fat value was significantly higher in fish fed diet containing 2.00%

methionine and was comparable to all the dietary groups except those receiving 1.00%

methionine, which exhibited significantly (P<0.05) lower fat value. Fish fed diet

containing 1.00% methionine had highest protein deposition value which was

comparable to that of 1.25% methionine level. Lowest protein deposition value was

recorded for fish receiving diet containing 2.00% methionine, which was

insignificantly different (P>0.05) from fish fed diet containing 0.5% methionine,

intermediate values were recorded for rest of the dietary groups (Fig. 3). Minimum

amount of moisture (P<0.05) was recorded in fish group fed test diet containing

1.00% dietaiy methionine.

DISCUSSION

Methionine is an indispensable amino acid required by teirestrial vertebrates as well

as vaiious fish species for noiTnal growth and metabolic functions. This sulphur

containing amino acid is needed for the synthesis of other sulphur-containing

biochemicals. Dose-response experiment with increasing supplies of amino acid are

accepted in principle as a method for deteiinining dietaiy requirement (Cowey, 1995),

30

The present finding indicates that 1.00 % of dietary methionine in presence of 1.00%

cystine is the optimum requirement for growth of fingerling Cirrhinus mrigala. Best

feed efficiency at this level of dietary methionine expressed as feed conversion ratio

was a reflection of highest weight gain, specific growth rate and protein efficiency

ratio. Broken-line regression analysis of the growth response curve at 1.00% dietaiy

methionine level (Fig. 1) indicates that increasing methionine beyond this level could

not provide significant additional growth. The break-point obtained in the growth

cui-ve at 1.00% methionine level further substantiate the 1.00% dietary methionine as

the optimum dietary requirement for this species. This value conesponding to 2.50 %>

(1.00 % cystine fixed) of dietaiy protein is veiy close to the requirement reported for

milkfish 2.50 % (0.75 % cystine fixed) of dietaiy protein (Borlongan and Coloso,

1993) and slightly lower then the requirement reported for other Indian major carp,

rohu 2.65 % (1.00 % cystine fixed) of dietary protein (Khan and Jafri, 1993) and

higher than the requirement reported for sea bass 2.20 % (1.00 % cystine fixed) of

dietary protein (Theabult et ai, 1985), chinook salmon 1.50 %> (1.00 % cystine fixed )

of dietary protein (Halver et al, 1959).

Methionine or total sulphur amino acids requirements vary from 2.20%) of

dietaiy protein to 6.50%o of dietaiy protein (DeSilva and Anderson, 1995). The

methionine requirement of fingerling Cirrhinus mrigala in presence of 1.00 % cystine

was determined to be 1.00 % of diet or 2.50 %> of dietaiy protein. Thus, based on the

above data the total sulphur amino acid requirement (methionine+cystine) was

determined to be 2.00 % of dry diet and 5.00 %> of dietary protein. This apparent

requirement was lower than the requirement reported for other Indian major carp, rohu

5.59 % of dietary protein (Khan and Jafri, 1993) and higher than the requirement

reported for milkfish 4.80%o (Borlongan and Coloso, 1993) chinook salmon 4.00%)

(Halver, 1959) sea bream 4.00 % (Luquet and Sabaut, 1974), channel catfish 2.34 %

(Harding et ai, 1977); 3.41 % (Cai and Burtle, 1996), common carp 3.10 % (Nose,

1979); 3.17 % (Schwarz et ai, 1998), Japanese eel 3.20 % (Nose, 1979), rainbow

trout 2.70 %) (Ogino, 1980); 2.20 %) (Walton et al, 1982 and 2.90%o Kim et al, 1992),

31

Mossambique tilapia 3.20 % (Jackson and Capper, 1982); 0.99 % (Jauncey et al,

1983), Nile tilapia 3.10 % (Santiago and Lovell, 1988), juvenile red drum 3.03 %

(Moon and Gatlin, 1991), Indian major cai-p, catla 3.75 % ( Ravi and Devaiaj, 1991),

juvenile hybrid striped bass 2.90 % (Keembiyehtty and Gatlin, 1993), Indian major

cai-p, rohu 3.23 % (Murthy and Varghese, 1998), African catfish 3.20 % (Fagbenro et

al, 1998), arctic chair 1.76 % (Simmons et al., 1999), juvenile Asian sea bass 2.90 %

(Coloso et al., 1999), juvenile yellow perch 3.10 % (Twibell et al, 2000), and

Japanese flounder, 3.10% (Alam et al., 2001).

Fish have a total sulphur amino acid requirement rather than a specific

methionine requirement (Wilson and Halver, 1986). The presence of dispensable

amino acid cystine in the diet represents a sparing effect in that it reduces the need for

methionine. Adequate amount of dietary cystine spares methionine from cystine

synthesis. Thus, the requirement for total sulphur amino acid can be met by either

methioiune alone or the proper mixture of methionine and cystine. Since the

requirement of methionine is influenced by the dietary cystine, it is determined at two

levels of cystine, presence or absence of cystine in the diet. Nose (1979) reported that

in the absence of cystine maximum growth rate was observed at 1.20 % methionine

level whereas in presence of 2.00 % cystine the fish fed on the diet with 0.80 %

methionine level showed optimal growth. Aiai et al., (as quoted by Nose 1979)

suggested that eel were found to require 1.20 % methionine in the diet in the absence

of cystine and 0.90 % with 1.00 % cystine in the diet. These results suggest that

requirement of caip and eel resembles each other. The present finding is almost

similar to these results. Harding et al., (1977) reported that methionine requirement of

channel catfish in the absence of cystine is about 0.56 % of diet or 2.34 % of dietary

protein. The values expressed as the per cent protein is lower than that of carp. Walton

et al., (1982) reported that 0.50 % methionine was adequate for 25 g rainbow trout,

when dietary cystine level was fixed at 2.00 % in the diet whereas the methionine

requirement was between 0.50-1.00 % of dry diet in the absence of cystine. Schwarz

et al., (1998) reported that methionine requirement of common caip weighing 569 g

32

was found to be 2.13% of the dietaiy protein in presence of 0.42 % of cystine of the

diy diet. This value seems to be quite low compared to the requirement level obtained

in present study. This decrease in requirement in large size fish points to the fact that

dietaiy methionine requirement decreases with advancing size/age of the fish.

It has been emphasized that pH neutialization of amino acids diet is necessaiy

(Nose, 1974), because caip do not have an acidic stomach and digestion proceeds in a

neutial or slightly alkaline environments. The consumption of an acidic amino acid

diet with a stiong buffering capacity might disturb the noimal assimilation of the

ingested amino acids. Murai el al., (198!) suggested that caip fingerlings fed gelatin

diet, supplemented with essential amino acids coated with casein, and casein-gelatin

contiol diet produced a faster growth than fish fed all ciystalline amino acids in the

diet, and also mentioned that amino acid coating by casein alters the relative

absoiption rate of certain amino acids in the gut. Additional evidence in support of

this hypothesis was obtained by again Murai et al., (1982) who compared the change

in the plasma free amino acids after feeding diets containing casein coated and

uncoated amino acids. Khan and Jafri (1993) pointed out that in comparison to all

ciystalline amino acid diets, casein-gelatin coated diet indicated improved utilization

of ciystalline amino acids in Laheo rohila, presumably through increased retention in

the gut. Hence, in the present study dietaiy methionine requirement of Cirrhinus

mrigala was detennined using neuti-alized (6N NaoH) casein-gelatin based amino

acids diets.

The wide variation obsei-ved in the methionine requirement among species may

be due to fish size, age, laboratory condition including feeding regime, feed

allowance, water temperature, stock density and ingredients used for basal diet such as

casein, gelatin, zein, glutin, fishmeal, soyabean meal and ciystalline amino acids in

various combinations. Although the value are similar for most of the species studied,

some variations exist perhaps due to species, the composition of the test diet, presence

or absence of added cystine and amount of other ciystalline amino acids used (Coloso

etal, 1999).

33

In the present study, excess amount of mthionine+cystine (total sulphur amino

acid) caused depressive effect such as reduction in growth and feed efficiency. Similar

obsei-vation was also reported for the Indian major carp, catla (Ravi and Devaraj,

1991), milkfish (Borlongan and Coloso, 1993) and rainbow trout (Kaushik and

Luquet, 1980). Dietaiy amino acids requirements is reported to be influenced by

feeding levels adopted (Chiu et ai, 1988). Digestibility, amino acids profile and

energy content bring about variable effects in amino acid requirement studies

(Simmons et ai, 1999 and De Silva el ai, 2000). Toxicity due to excessive intake of

one amino acid results in poor growth because disproportionate amount of one amino

acid affect the utilization of the other amino acids (Coloso et al., 1999). Reduction in

the growth rate in fish fed diets with higher level of methionine+cystine may be due

to toxic effect (Choo et al., 1991) and stiess caused by excess amount of total sulphur

amino acid in the body of the fish leading to extra energy expenditure toward

deamination and excretion of the same (Walton et ai, 1985). Except for poor growth

and low feed efficiency, which resulted in depressed growth, no pathological

symptoms were obsei-ved in mrigala fmgerling fed low levels of dietary methionine.

The data generated in the present study on total sulphur amino acid requirement of

fmgerling Cirrhinus mrigala as 2.00% (1.00% cystine) of the dry diet would be useful

in developing sulphur amino acid balanced diets for intensive culture of this species.

SUMMARY

Indian major caip, Cirrhinus mrigala fingerling (3.85± 0.50 cm, 0.50± 0.02 g) were

fed isonitiogenous and isocaloric diets (40% CP, 4.28 kcal/100 g\ GE) containing

casein, gelatin and ciystalline amino acids with graded levels of L- methionine (0.50,

0.75, 1.00, 1.25, 1.50 and 2.00 g/ 100 g, diy diet) with 1.00% cystine fixed, to

determine its dietaiy methionine requirement. Feeding trial was conducted in tiiplicate

for six weeks. Diets were ftd twice a day at 0800 and 1600 h at 5% of body

34

weight/day. The ration size and feeding regime were worked out prior to the stait of

the feeding tiial. Weight gain (158%) and feed conversion ratio (1.45) were

significantly (P<0.05) higher in fish fed diet containing 1.00% methionine with 1.00%

cystine fixed. Broken line regression analysis of the weight gain data also indicated

the dietaiy methionine requirement to be l.OOg/ lOOg of diy diet, corresponding to

2.50% of dietaiy protein. Second-degree polynomial regression analysis was

employed to detennine the relationship between food conversion ratio (FCR) and

dietaiy methionine levels which also indicated that the best FCR occuned at

approximately 1.09% dietaiy methionine level. Carcass composition offish fed diet

containing graded levels of methionine varied significantly (P<0.05) except carcass

ash content which showed insignificant (P>0.05) differences among the dietary

methionine levels. Water temperatme, dissolved oxygen, dissolved free carbon

dioxide, pH and total alkalinity were checked regularly during the experiment. No

mortality was obsei-ved during the length of the feed trial.

35

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^' • — '

rt

cn 0 )

+i 0 ^ '^

ro

r-'—' +i 0 rn ^

^

+i m 0 \

c^

JD 1 / ^

^ +i 0 >n c^

0 r i

+i ro 0

•=t'

« cn 1—1

+i 0 (N (N

!fl 1 '

^ + i • ' (•n

(N

rt 00 0

+i ^ 0

r-i

f ^

r-0

+i >n ' . '^

^ 0

'—' +i 0 0

( N '

m C-A

+\ 00 r--r-' r~~

0^

+i 00 00

0 00

•vO

'—' +\ o\ 1—1

m '

00

+i en ^ - H

m' '—'

KJ

rn •—' +i ( N ( N

^'

i n

+1 ( 0

f o

d ( N

^ +i r—' ( N

(N'

ON 0

+i <N ^ rn

3

O X M

<

o o A a .

c

- a _>>

c O

'S

c

a-

2 w on +1 en

1 , y

"cL

m «+-! 0 on

P

> ^ s (D

o; 0. D C/1

 c CIj (U

Fig. 1. Relationship between live-weight gain per cent and dietary methionine levels.

0,75 1.00 1.25 1.50 1.75

METHIONINE LEVEL ( 'I. d ry diet )

Fig. 1.

Fig. 2. Second-order polynomial relationship of food conversion ratio to methionine

levels.

o o Q O O

1.5

Y = 2,781X - 6.7215X ^ 5.562 {r=0.1694)

1 0,46 0.76 1.06 1,36 1,66

METHIONINE LEVEL (% dry diet)

1,96

Fig. 2.

Fig. 3. Relationship between protein deposition and dietaiy methionine levels.

0.5 0.75 1 1.25

METHIONINE LEVEL (%dry diet)

1.5

Fig.3.

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