FEEDING EXPERIMENT STUDIES
Pairat Kosutarak, Ph.D.
Inland Feed Research Institute, Inland Fisheries Research and Development Bureau,
Department of Fisheries
To make money in aquaculture, transforming feed to food must be done efficiency and
economically. Aquatic animals can do this, but the principles of nutrition must be applied.
Successful nutritional practices also depend on breeding, health and management.
Feed can be designed to meet any of a number of goals, including rapid growth for
market, successful reproduction, or low pollution in the hatchery effluent. The knowledge in
nutrition is essential, because it influences the way in which feed ingredients are chosen,
prepared, combined, and processed into feeds.
In recent years, advances in feed pelleting methods, changes in the availability and
quality of ingredients used in feeds, development of new feed ingredients, cultivation of new
aquatic species, and advances in the knowledge of aquaculture nutritional requirements have
resulted especially in feed manufacturing for aquaculture. However, the knowledge of
aquaculture nutrition has lagged behind that of other farmed animals (chicken, pig, castle
etc.). Basically studies are similar to land animals (growth, reproduction, physiological
functions etc.) and generally classified as proteins, carbohydrates, lipids, vitamins and
minerals. Information on the nutrition of aquatic animals is important for promoting the
aquaculture business. Thus feeding experiments are the most useful method in obtaining
results.
Outline of Feeding Experiment
Planing (design of experiment)
Checking ingredients (available or not)
Diet preparation for preculture
Collecting test animals
Preculture & Setting the rearing units etc.
Selection of test animals
Diet preparation for feeding experiment
Start experiment
Daily/intermediate measurement
Terminate experiment
Advanced Freshwater Aquaculture: Feeding Experiment Studies
188
Evaluation of results of feeding experiments
There are several parameters and measurements have been involved in feeding
experiments. Thus it is important to plan which parameters and measurements that possible to
investigate. Because not everyone will have equipments/facilities/budget etc.
I. Parameters on nutritive value of diets
The following parameters are determined for evaluation of nutritive values of diets:
1. Proximate analysis: crude protein, moisture, fiber, fat, ash, and nitrogen-free extract
(NFE)
2. Fatty acids composition
3. Amino acids composition
4. Analysis of test nutrients
II. Parameters on test animals
The following parameters are calculated for evaluation of test animalsperformances
1.
Daily feed consumption
Feed intake (g)
Daily feed consumption (%) =
X 100
Initial body wt.+Final body wt.
2
2.
Percent gain (Growth rate)
Percent gain (%)
=
3.
Specific growth rate (SGR)
SGR (%/day)
=
ln(Final body wt.)-ln(Initial body wt.)
(Final body wt.-Initial body wt.)
X 100
Initial body wt.
Feeding period (day)
4.
5.
6.
7.
X day
X 100
Mortality
Mortality is expressed as number or percent of dead animals per unit
time.
Morbidity
Morbidity is expressed as number or percent of dead animals due to the
disease/ill per unit time.
Body composition
Proximate analysis and energy content of whole body or tissues are
recommended for the initial and final samples.
Health status
Health status is possibly evaluated by:
- Histological examination (blood tissues)
- Specific comments for high mortality from bacteria, fungal and
virus disease, parasitic organisms
- External and internal appearances
- Condition factor (Fatness)
Body wt. (g)
Condition factor (%)
=
X 100
Total length3 (cm)
- Hepatosomatic index (HSI)
Liver wt.
HSI (%)
=
X 100
Body wt.
Advanced Freshwater Aquaculture: Feeding Experiment Studies
189
III. Parameters on feed utilization
The following parameters are calculated for evaluation of feed utilization :
1.
Feed conversion rate (FCR)
Dry weight of feed consumed
FCR =
Wet weight gain
2.
Protein efficiency ratio (PER)
Wet weight gain
PER =
Amount protein consumed (dry basis)
3.
4.
5.
6.
Energy efficiency ratio (EER)
Wet weight gain
EER =
Amount energy consumed (dry basis)
Protein deposition
Protein deposition (g)
= (final ind. wt x DM final carcass x protein composition final carcass)
–
(initial ind. wt x DM initial carcass x protein composition initial carcass)
Lipid deposition
Lipid deposition (g)
= (final ind. wt x DM final carcass x lipid composition final carcass)
–
(initial ind. wt. x DM initial carcass x lipid composition initial carcass)
Protein retention efficiency
Protein retention efficiency (%)
Individual protein deposition (dry basis)
X 100
Individual protein consumption (dry basis)
=
7.
Lipid retention efficiency
Lipid retention efficiency
Individual lipid deposition (dry basis)
Individual lipid consumption (dry basis)
=
X 100
LIPIDS
GENERAL INTRODUCTION
Lipids are organic compounds found in plant and animals tissues, and are oily or
greasy substances soluble in organic solvents such as benzene, ether or chloroform, but only
sparingly soluble in water. There are different classes of lipids which are listed in table 1.
Table 1. Classification of lipids
Simple
Fats (TG)
Waxes
Saponifiable
Compound (Polar lipids)
Phospholipids (PL)
Sphingolipids
Glycolipids
Lipoproteins
Non-saponifiable
Steroids
Carotinoids
Fat-soluble vitamin
Advanced Freshwater Aquaculture: Feeding Experiment Studies
190
From the viewpoint of the amounts present in fish body and their feeds, the neutral fats
are by far the most important members of the lipid groups, but the other lipids such as PL play
very significant roles in nutrition and in physiology.
1) Fats (Triglycerides)
Fats are important sources of stored energy in plants and animals as well, and are
characterized by their high energy value. One gram of fat yeilds 9.3 kcal of heat when
completely combusted compared with the trihydric alcohol glycerol, and are called
triglycerides (TG).
Fatty acids are long-chain organic acids, having 4-24 carbon atoms (mostly 16 or 18)
and a single carboxyl group. The same or different fatty acids may be in all three position.
The chain length and degree of unsaturation of the fatty acids making up the TG determine its
physical and chemical properties. TG of saturated fatty acids (SFA) containing at least ten
carbon atoms are solid at room temperature, whereas those with less ten carbon atoms are
liquids. In oils the percentage of unsaturated long-chain fatty acids surpasses that of SFA.
The acid found in fats usually have an even number of carbon atoms, which is to be
expected in view of their mode of biosynthesis.
The fatty acid composition of fats of animal and plant origin is given in table 2 and 3.
The composition of fats present in the animal body depends on the species of animal, and
varies in the different tissues; also it is influenced to some extent by the diet.
Table 2. Composition (%) of fat in some animal species
Saturated fatty
acids
C14 and
lees
Fish oil
Chicken fat
Pork fat
Butter
Beef fat
15
C16+C18
11-15
25
35
30-40
50
Unsaturated fatty acids
Linoleic
+
Linolenic
acid
20
25-30
5-7
0.50
0.5
Oleic
acid
40
50
35
35
C20 and
more
50
Consistency
Liquid
Soft
Soft
Soft
Solid
Table 3. Composition (%) of fat in seeds
Linseed oil
Soybean oil
Sunflower
seed oil
Coconut oil
Cottonseed oil
Saturated fatty
acids
C14 and
C16+C18
less
6-16
7-10
6-15
80
11
24-29
Unsaturated fatty acids
13-36
23-30
20-50
Linoleic
acid
10-25
50-60
30-60
5-7
15-20
49-57
Oleic acid
Linoleic
acid
30-50
5-9
Advanced Freshwater Aquaculture: Feeding Experiment Studies
Consisten
cy
Liquid
Liquid
Liquid
Solid
Liquid
191
2) Phospholipids (Phosphatides)
Phospholipids (PL) are esters of glycerol in which two of the hydroxyl groups of
glycerol are esterified by long-chain fatty acids. The third is esterified by phosphoric acid.
The most commonly occurring PL in animals and plants are the lecithins in which the
phosphoric acid is also esterified by the nitrogen base choline.
Due to the present of the hydrophilic phosphate group and the hydrophobic fatty acid
chains within the same molecule, the PL exert emulsifying properties and fulfil important
functions in lipid transport in blood and as components of animal cell membranes. PL are
abundant in heart, kidney and nervous tissues. The PL in these tissues are higher in
unsaturated fatty acids than TG of adipose tissue. The lipids of soybean seeds also contain
lecithin. Lecithin isolated from soybeans is used as an emulsifying agent in milk substitutes
for claves.
IMPORTANT ROLES OF LIPIDS
Dietary lipids play important roles in the energy production processes of animal
tissues and as the source of EFA. Besides these functions they do have other important dietary
roles as carriers of certain non-fat nutrients, notably the fat-soluble vitamins A, D, E and K. In
the carnivorous fish such as rainbow trout, eel, yellowtail and plaice which have limited
ability to utilize carbohydrates of high molecular weight as an energy source (8-9 kcal/ g)
dietary lipids play an important role in this respect and have a sparing action on dietary
protein.
Researches about fish nutrition related to the development of aquaculture early
focused on dietary requirement for lipids as it has been early reported that inadequate supply
of dietary lipids lead to poor growth and pathological manifestations.
Animals are requiring dietary lipids, first as a source of metabolic energy [formation
of ATP (adenosine triphosphate) through β-oxidation of TG)], secondly to maintain the
structure and functions of cellular membranes, mainly built of PL, and thirdly to synthesize
hormones, like the prostaglandines, and to carry nutrients, like the fat soluble vitamins. Major
differences between lipids of fish and land animals can be summarize as follow.
1) Lipids play the first role as source of metabolic energy in fish, which have a low
ability to use energetically glucids, in contrast to land animals.
2) Fish tissue lipids, specially PL of the biomembranes, are composed of large
amounts of highly unsaturated fatty acids (HUFA), rarely recorded in land animals tissue .The
fatty acids are predominantly of the n-3 serie in fish, whereas they are of the n-6 serie in
terrestrial animals.
Tissue fatty acids are coming from two sources, firstly from the supplied diet and
secondly from the organism biosynthesis.
CLASSICFICATION OF FATTY ACIDS
Fatty acids will be considered as four classes:
1) Saturated fatty acids(SFA)
SFA vary in chain length from 4 to 18 carbons and the major saturated fatty acids in
the diet are lauric (12:0), myristic (14:0), palmitic (16:0), and stearic (18:0) acids. The major
dietary sources of SFA are animals products (meat, and dairy products), tropical oils (coconut,
palm, and palm kernel oils) added to processed foods, and hydrogenated vegetable oils.
2) Monounsaturated fatty acids (MUFA)
The primary MUFA in the diet are palmitoleic(16:1n-9) and oleic (18:1n-9) acids;
however, for practical purposes this class of fatty acids is represented by oleic acids, which is
found in olive oil, rapeseed oil (canola oil), cocoa-butter, and beef.
3),4) Polyunsaturated fatty acids (PUFA)
There are two major classes of PUFA:n-3 fatty acids, such as eicosapentaenoic
Advanced Freshwater Aquaculture: Feeding Experiment Studies
192
(20:5n-3; EPA) and docosahexaenoic (22:6n-3; DHA) acids, found in fish oils and as minor
constituents of some vegetable oils; and n-6 fatty acids, including linoleic acids (18:2n-6),
found in vegetable oils such as corn, cottonseed, and soybean oils.
In some literatures of aquaculture nutrition, n-3 highlyunsaturated fatty acids
(n-3 HUFA) is referred to n-3 PUFA which have ≥ 20 carbons with 4, 5 and 6 double bonds;
especially, 20:5 n-3(EPA) and 22:6 n-3 (DHA)
DESATURATION AND ELONGATION OF FATTY ACIDS
Fish ingest an abundance of unsaturated fatty acids which can be incorporated directly
into body lipids and are also capable of modifying both dietary fatty acids and the fatty acid
products of endogenous synthesis by desaturation and elongation. Fish, like all other animals,
do not posses the desaturases necessary for the formation of 18:2n-6 (linoleic acid) and 18:3n3 (linolenic) from 18:1n-9 (oleic acid). Consequently, all n-3 and n-6 PUFA in fish lipids
ultimately originate from n-3 and n-6 PUFA formed in plants.
ESSENTIAL FATTY ACIDS REQUIREMENT OF FISH
Dietary lipids provide those PUFA which fish, like all animals, cannot synthesize de
novo but require for the maintenance of cellular function – the EFA. Despite the abundance of
n-3 PUFA in natural fish oils, early fish nutritionists assumed the EFA of fish to be the same
as those of land animals and used vegetable oils rich in 18:2n-6 as dietary lipids, with poor
results. Numerous studies eventually established the requirement of fish in general for the n-3
PUFA but the extent to which the n-6 series are EFA remains to be established.
The nutritional aspects of EFA in fish have been extensively studies. Several studies
have indicates that the EFA requirements of fish differ considerably from species to species
and will vary depending on the percentage of dietary lipid.
Generally, the pattern of EFA requirements for aquatic animals are classified into
several groups; freshwater fish (cold temperature), freshwater fish (mild temperature), marine
fish-, and tilapia (tropical) types. The difference in EFA requirements among fish species are
thought to relate closely to the discrepancy in ability for bioconversion of dietary n-3 C18 fatty
acids to n-3 HUFA:C20 and C22 among these fish. Except for the tilapia species, n-3 HUFA
have high EFA efficacy for most fish and crustaceans.
EFA DEFICIENCY SIGNS
Two signs common to all species of EFA-deficient fish are poor growth rate and low
feed conversion. These signs have been demonstrated in feeding studies with several species
of freshwater fish, including rainbow trout, salmon, eel, tilapia and carp, and with marine
species such as red sea bream and turbot.
n-3 HUFA AS EFA
Several studies have demonstrated that fish liver oil including the residual oil from
molecular distillation of pollock liver oil for production of vitamin concentrates, were very
effective in enhancing growth and improving feed conversion in many fish species. The
mechanism of this growth stimulating effect is unknown but earlier workers suggested that it
could be attributable to:
1) fat – soluble vitamin A and D
2) phospholipids
3) polyunsaturated fatty acids, in pollock liver oil. The later results obtained on EFA
requirements of fish, however, suggested that the nutritive value of dietary pollock liver oil in
fish was probably due to PUFA, especially n-3 HUFA (such as 20:5n-3 and 22:6n-3), in the
fatty acid fraction.
The EFA of n-3 HUFA was examined in many fish species. The requirement of n-3
HUFA for some species of marine finfish as shown in Table 4.
Advanced Freshwater Aquaculture: Feeding Experiment Studies
193
Table 4. Requirements of n-3 HUFA for marine finfish.
Fish species
n-3 HUFA (%)1
Red sea bream (Pagrus major)
0.5
Yellowtail (Seriola quinqueradiata)
2.0
Striped jack (Pseudocaranx dentex )
1.7
Striped knifejaw (Oplegnathus fasciatus)
1.0
Flounder (Paralichthys fasciatus)
2.0
Seabass (Lates calcalifer)
1.0
Turbot (Scophthalmus maximus)
0.8
Gilthead seabream (Sparus aurata)
1.0
1
Requirement changes according to the grown stages and experimental conditions.
OXIDATION OF LIPIDS
1) Factors Affecting Fish Lipid Oxidation
The factors which influence lipid oxidation in fish include the fatty acid composition
of lipids, their disposition, the presence or absence of activators and inhibitors (heme, metal,
ions, pH value, oxidative enzymes, tocopherol, carotenoids), and external factors such as
storage temperature, time, light, oxygen pressure, water activity, and packing conditions.
The type of fatty acid present is a major factor in determining the oxidative stability of
lipids. In general, the rate of autoxidation increase as the number of double bonds increase.
2) Toxic of Oxidized Fats and Oils
Three distinct classes of compounds detected in oxidized fats and oils have been
shown to have toxic effects
1) fatty acid hydroperoxides and their end products,
2) polymeric material and
3) oxidized sterols
the feeding of highly oxidized fats, in the form of an oxidized cod liver oil, to animals
has produced a wide spectrum of injurious effects including diarrhoea, loss of appetite,
growth retardation, cardiomyopathy [ disease of the heart muscle (myocardium)],
hepatomegaly (enlargement of the liver), haemolytic anaemia (anaemia due to an increase in
the rate of destruction of circulating erythrocytes) and accumulation of peroxides in adipose
tissue.
There is a large number of antioxidants available. Antioxidants for fats and oils may
be arbitrarily divided into two groups: primary antioxidants, and synergists. The first group
includes those which are effective in a very small amounts; these in general are characterized
by being hydroxyl or nitrogen derivates of aromatic compounds, and are free-radical
acceptors. Those antioxidants which are permitted for use in United States are shown in Table
5.
Advanced Freshwater Aquaculture: Feeding Experiment Studies
194
Table 5. Partial list of food additives permitted in United States*
Antioxidants
Tocopherols
Butylated hydroxyanisole (BHA)
Butylated hydroxytoluene (BHT)
Propyl gallate (PG)
Nordihydroguaretic acid (NDGA)
Gum guaiac
Thiodipropionic acid
Dilauryl thiodipropionate
Synergists
Ascorbic acid
Erythobic acid
Ascorbyl palmitate
Citric acid
Isopropyl citrate
Stearoyl citrate
Lecithin
Phosphoric acid
* Usually not more than 0.02%
In commercial situations, diets often contain high levels of PUFA from fish oil and are
prepared by steam pelleting and stored in paper bags at room temperature for long periods of
time. Thus synthetic antioxidants, such as BHT, BHA, and ethoxyquin, are commonly used in
animal feeds to reduce oxidative rancidity. These synthetic antioxidants exhibit some vitamin
E activity and may spare dietary vitamin E when present in diet
SUGGESTED READING
JICA (Japan International Cooperation Agency). 1988. Fish nutrition and mariculture.
JICA Textbook) The general aquaculture Course, edited by T. Watanabe. 233 p.
Halver, J.E. 1989. Fish nutrition. Second edition, Academic Press, New York. 798 p.
Gurr, M.I. and Harwood, J.L. 1991. Lipid biochemistry. An introduction. Fourth edition.
Chapman & Hall. 406 p.
Advanced Freshwater Aquaculture: Feeding Experiment Studies