2-2014 9.03biological and Microbiological Evaluation of Aquafeeds and feedstuffs-MBT PDF

Myrna B. Teruel, Ph.
D
Scientist
Aquaculture Department
Southeast Asian Fisheries Development Center
Tigbauan, Iloilo 5021 PHILIPPINES
*Lecture notes submitted to Technology and Information Division for the Training Course on
Feed Formulation and Feed Evaluation for Aquaculture Species, September 1-5, 2014
Introduction
the use of quality feeds is important in the success of
any aquaculture venture since 50-60% of the cost of production is
mainly attributed to feeds.
feed quality is highly dependent on the quality of raw material
and the processing technique.
formulated feed that makes use of low quality raw materials
will not give the fish farmer any significant benefit. Feedstuffs and
finished feeds should, therefore, undergo the process of evaluation
and quality control in order to produce high quality feed.
systematic evaluation of feedstuffs and feed using

physical, chemical, microbiological, and biological methods
is necessary to assure their effectiveness when fed to fish.
this procedure starts from procurement of feedstuffs and
continues to feed processing until manufacture and storage
of the finished product.
the finished feed must contain all the nutrients required
by the fish in adequate amounts and proper proportions.
growth response of animals to feed quality.
High quality feed (1) = Healthy and bigger fish.
Low quality feed (2) = Stunted fish.
Feed and ingredient evaluations

defined ???
This is a step-wise process in which feeds and feed
ingredients are analyzed by a series of physical , chemical,

microbiological and biological tests, blended to produce an
assessment to ensure that the final product is of high
quality and that deleterious effects of the produce are avoided.
No single test will provide the necessary data for adequate
feed evaluation. Hence, a variety of evaluation/assessment

methods are utilized to provide information required to
assess quality and nutritional value of feeds and feed
ingredients.
Evaluation of Feedstuffs and Aquafeeds

Four methods of feed and feedstuffs evaluation
1. Physical
- detects the presence of adulterants in feedstuffs.
2. Chemical
- quantifies the amount of given compound present in the feed and
feedstuffs.
3. Microbiological
- involves the use of microorganisms to evaluate the
quality of the feed or the feed ingredient.
4. Biological
- involves actual feeding experiment. Gives an
accurate estimate of feed utilization
Methods of Feed/Ingredient Evaluation

Approaches
Advantages
In vivo approaches
On-farm experiment
Reliable
Growth/digestibility
Reliable
Disadvantages
Costly; Experimental
designs limitations
Costly; limited accessi
bility
Lab approaches
In vitro assays
Proximate analyses
Fast; reliable
Fast, cheap, easy
High development cost
Analytical
errors/limitations
3. Microbiological Evaluation
a method of feed/feed ingredients evaluation that
utilizes various microorganisms (e.g. Lactobacillus

rhamnosus; Enterococcus hirae; Pediococcus
acidilactica) in order to determine the nutritional
quality of a product.
Amino acid composition
this method is valuable in analyzing mixtures
of amino acids because of the speed and
reproducibility of results obtained.
a nutrient medium which contains all the
essential compounds needed for the growth
of a particular microorganism except the
amino acid to be assayed is prepared.
Lactobacilli
the addition of this amino acid results in the growth of
the microorganism in proportion to the amount of amino acid
added. Culture tubes are set up and graded amounts of the
unknown are added to a series of tubes.
standards are set at the same time with graded amounts of the
pure amino acid. The unknown can be compared with standards by
measuring the rate of growth of the microorganism.
with organisms that form acid such as Lactobacilli , titration of
the acid formed can be used as a measure of the number of cells
present. Pure cultures need to be used in this
kind of evaluation.
a. Determining amino acid composition
rapid development of microbiological methods for
amino acid determination

has greatly facilitated their application to the
estimation of amino acids in feeds and feed
ingredients.
considerable data are now available to show that these
techniques can be utilized
determine the amino acid composition of feeds and feed
ingredients following hydrolysis without removal of fat,
water or carbohydrate material.
3. Microbiological evaluation
Procedure:
Feed samples obtained by random sampling of the feeds
and feed ingredients (e. g. corn, corn gluten meal, meat
and bone scraps, soybean oil, soybean meal, alfalfa leaf
meal)are finely ground prior to assay to assure
homogeneity.
L. 'arabinosus is used as the test organism for amino
acid determinations.
Pure amino acid standards are used to eliminate
possible source of error.
3. Microbiological Determination
Express amino acid values on the basis of 10% activity for the 1-
isomer and no activity for the d-isomer.
Hydrolyze samples by autoclaving with 2N HCl at 15 Ibs.
pressure for 10 hours for maximum liberation of amino acids.

After hydrolysis, neutralize samples and take aliquots taken for
assay.
Express results as per cent of each amino acid in the sample as
analyzed and as per cent in the crude protein (calculated to 16%

nitrogen).
3 purified proteins (casein, egg albumin and gelatin) are usually
used as standards.
Amino acid content of rations determined by actual
analysis and calculated from the values obtained for the

components. All values are expressed as per cent in the
ration.
Leucine
No.1 (determined) 2.49

No.1 (calculated) 2.54
No. 2 (determined) 2.47
No. 2 (calculated) 2.64
Valine
Isoleucine
Phenylalanine
1.17
1.38
1.27
1.42
1.06
1.10
1.10
1.21
1.07
1.14
1.14
1.19
The amount of each amino acid contributed by the various

protein ingredients was calculated on the basis of the
percentage of the ration in the ingredient.
Microbiological Procedure for

Amino acids Assay
Applicable only to materials containing free forms of
amino acids in absence of appreciable amount of

protein
Stock Solutions for Basal Media

Stock Solutions for
Basal Media
Amino acid
solution
Adenin-guanineuracil solution
Salt Solution A
Salt Solution B
Vitamin Solution I
Vitamin Solution II
Basal Medium
Culture and
suspension media
In preparing
medium for given
assay, omit
particular amino
acid to be assayed
from medium
Agar culture
medium
Liquid culture
medium
Liquid suspension
medium
For detailed method for the preparation of stock solutions, refer to

AOAC 2000 960.47.
Stock cultures of test organisms

For use in assay of
isoleucine, leucine,
threonine,
tryptophan, valine,
arginine and
histidine
For use in assay of

isoleucine, leucine,
methionine,
phenylalanine,
tryptophan, and
valine
For use in assay of

lysine, methionine,
phenylalanine,
tyrosine, cystine
and histidine
Streptococcus
faecalis
Lactobacillus
plantarum
Pediococcus
cerevisiae
Inoculum
Suspend cells in 10 ml
suspension medium
Incubate (16-24 h, 34C)

Centrifuge and decant
Transfer of cells from stock
culture to 10 ml liquid
culture medium
For the reference standard

solutions of amino acids,
calibration of photometer
and preparation of test
samples refer to AOAC
2000 960.47.
Assay
Using working standard solutions, test solutions,
and basal medium (omitting amino acid being
assayed) perform assay, determination and

calculation as described in AOAC (2000) 960.47.
Microbiological Method of
Feed Evaluation
Vitamin Composition:
Vitamin B12 (Cobalamine)

Niacin (vitamin B3)
(nicotinic acid)
Folic acid (vitamin B9)
Microbiological Method of Feed

Evaluation
The microbial assay of vitamins is based upon the
comparison of the stimulation of growth of bacteria by

measured concentration of vitamin with that produced by
known concentration of standard preparation of vitamin
having known activity.
growth of microorganisms is proportional to requirement
for specific vitamin

The microbiological assay of vitamins utilizes various
strains of microbes.
Stock Cultures of Test Organisms

Lactobacillus leichmannii for use in assay of cobalamine
Lactobacillus plantarum for use in assay of niacin
Streptococcus faecalis for use in assay of folic acid
Prepare stab culture of appropriate test organism in 1 tubes of agar
culture medium.
Incubate 6-24 h at any selected temperature (30-40C) held constant to
within 0.5C.
Store in dark room at 10C
*Before using new culture in assay, make several successive transfers of

culture in 1-2 week period.
1. Vitamin B12 (Cobalamin)

Applicable to materials
containing 0.1 g of Vitamin B12

activity/g or ml
Refer to AOAC Official method
(2000) 952.20 for the preparation

of Basal Medium Stock Solution
and Cyanocobalamine Standard
Solutions (stock solution,
intermediate solution and
working solution).

A sufficient intake of vitamin B12, also known as
cobalamin, is important as it helps the body to convert

food into glucose, which is used to produce energy.
Cobalamine is stable to processing for short periods at
1210C but very unstable in the presence of light.

Processing of feeds/feed ingredients containing
cyanocobalamine should be done in the dark room.

Inoculum
1. Transfer
Lactobacillus
leichmanni to
sterile tube
containing 10 ml
liquid culture
medium
2. Incubate (624 h between 30

and 40C)
5. Wash cells with

0.9% NaCl soln
3. Centrifuge
culture
6. Resuspend cells
in 10 ml sterile
0.9% NaCl soln
4. decant
Test Solution (AOAC 2000)

Must contain 0.03 mg Na2S2O5/ml
7. Dilute aliquot
with the same soln
to give T equivalent
to that for dried cell
weight of 0.5- 0.75
mg/tube
Read against
suspension
medium set
at 100% T. cell
suspension so
obtained is
inoculum

Assay Tubes
To test tubes, add in
duplicate or triplicate
0.0 (for uninnoculated
blanks), 0.0 (for
inoculated blanks), 1.0,
2.0, 3.0, 4.0, and 5.0 ml,
respectively, of
standard solution)
Meticulously
cleanse by suitable
means followed by
heating 1-2 h at
250C
Incubate between 30
and 40C held constant
to within 0.5C
autoclave at 121123C reaching

this temperature
in 10 min.
add H2O and

5.0 ml
appropriate
basal medium
stock solution
and mix
Cool and inoculate each

tube, except 1 set of
duplicate (or replicate)
tubes containing 0.0 ml
standard solution
(uninoculated), with 1
drop appropriate
inoculum.

Titrimetric Test
1. Incubate tubes 72 h and then titrate contents of each tube with 0.1 M NaOH,
using bromothymol blue indicator, or to pH 6.8 measure potentiometrically.
* Disregard results of assay if response at inoculated blank level is equivalent to
titration of > 1.5 ml greater than that of uninoculated blank level.
* Response at 5.0 ml level of standard solution should be equivalent to titration of
8-12 ml.
2. Determine amount of vitamin for each level of test solution by interpolation
from standard curve.
* Discard any observed titration values equivalent to < 0.5 ml or > 4.5 ml,
respectively, of standard solution.
2. Niacin and Niacinamide (vitamin B6)

Applicable to all food products
Refer to AOAC Official method (2000) 944.13 for the
preparation of Basal Medium Stock Solution and

Niacin Standard Solutions (stock solution,
intermediate solution and working solution).
Niacin is sensitive to light and high temperature.
Niacin and Niacinamide

Inoculum
Liquid
culture
medium
1.Dilute basal
medium stock
solution with
aqueous solution
containing 0.2 g
niacin/ml
Inoculum
1. Transfer lactobacillus
plantarum to sterile
tube containing 10 ml
liquid culture medium
2. Incubate 6-24 h
(between 30 and 40 C
2. Add 10 ml of diluted
medium to test tubes
3. Centrifuge culture
and decant
supernatant
3. Autoclave (15 min,

121-123C)
4. Wash cells with 0.9%

NaCl soln
4. Cool and store in

dark at 10C
5. Resuspend cells in
0.9% NaCl soln
6. Cell suspension so
obtained is inoculum

Test Solution
preparation of test solution
Proceed to assay using standard solution, test
solution, basal medium stock solution and inoculum.

Assay Tubes
blanks), 0.0 (for
2.0, 3.0, 4.0, and 5.0 ml,
respectively, of
standard solution)
Meticulously
cleanse by suitable
means followed by
heating 1-2 h at
250C
Incubate between 30
to within 0.5C

this temperature
in 10 min.
add H2O and

5.0 ml
appropriate
basal medium
stock solution
and mix

standard solution
drop appropriate
inoculum.
Niacin (Vitamin B3)

Titrimetric Method
1. Incubate tubes 72 h and then titrate contents of each tube with 0.1 M
NaOH, using bromothymol blue indicator, or to pH 6.8 measure
potentiometrically.
* Disregard results of assay if response at inoculated blank level is
equivalent to titration of > 1.5 ml greater than that of uninoculated blank
level.
* Response at 5.0 ml level of standard solution should be equivalent to

titration of 8-12 ml.
Niacin (Vitamin B3)

2. Determine amount of vitamin for each level of test
solution by interpolation from standard curve.
* Discard any observed titration values equivalent to <
0.5 ml or > 4.5 ml, respectively, of standard solution.
The value so obtained is potency of sample expressed as
niacin.
Multiply the value by 0.992 if potency is to be expressed as
niacinamide.
3. Folic acid (Vitamin B9)

Applicable only to materials containing free forms of
folic acid.
preparation of Basal Medium Stock Solution and Folic

acid Standard Solutions (stock solution, intermediate
solution I and II and working solution).
Folic acid (Vitamin B9)

Inoculum
Transfer Streptococcus faecalis to sterile tube containing 10 ml liquid
culture medium
Incubate 6-24 h (between 30 and 40C)
Centrifuge culture and decant supernatant
Wash cell with 0.9% NaCl solution
Resuspend cells in 10 ml 0.9% NaCl solution
Cell suspension so obtained is inoculum
Test Solution
Refer to AOAC (2000)Official Method 944.12 for the preparation of test
solution.
Perform assay using standard solution, test solution, basal medium stock
solution and inoculum.
Folic Acid (Vitamin B9)

Assay Tubes
blanks), 0.0 (for
2.0, 3.0, 4.0, and 5.0 ml,
respectively, of
standard solution)
Meticulously
cleanse by suitable
means followed by
heating 1-2 h at
250C
Incubate between 30
to within 0.5C

this temperature
in 10 min.
add H2O and

5.0 ml
appropriate
basal medium
stock solution
and mix

standard solution
drop appropriate
inoculum.
Folic acid (Vitamin B9)

Titrimetric Method
1. Incubate tubes 72 h and then titrate contents of each tube with 0.1 M NaOH,
using bromothymol blue indicator, or to pH 6.8 measure potentiometrically.
* Disregard results of assay if response at inoculated blank level is equivalent to
titration of > 1.5 ml greater than that of uninoculated blank level.
* Response at 5.0 ml level of standard solution should be equivalent to titration of
8-12 ml.
2. Determine amount of vitamin for each level of test solution by interpolation
from standard curve.
* Discard any observed titration values equivalent to < 0.5 ml or > 4.5 ml,
respectively, of standard solution.
Calculations
For each level of test solution used, calculate vitamin
content/ml of test solution.

Calculate average of values obtained from tubes that do not
vary by 10% from this average.
If number of acceptable values remaining is < 2/3 of original
number of tubes used in the 4 levels of test solution, data are
insufficient for calculating potency of product.
If number of acceptable values remaining is 2/3 of original
number of tubes, calculate potency of product from average of
them.
Animal feeds
Aquatic animals reared intensively require large amounts of plant or
animal protein in the feed. This material is prepared from meat,

offal, bones, blood or feathers, or combinations. Animal
proteins often contain high level of Salmonella which depends
on the initial contamination of the raw materials and on the
hygiene of feed manufacture.
Animals fed with contaminated feed, often carry these salmonella in
their intestinal tracts, with no sign of illness.
Feed from infected ingredients may become contaminated during
processing and feed preparation (inadequate cooking and

storage procedures).
What is salmonella?
Salmonella is bacteria that can cause a gastrointestinal
infection known as salmonellosis. Usually

salmonellosis is referred to as "salmonella." This
infection can occur in humans and animals.
People become infected with salmonella by swallowing
the bacterium. This can happen when contaminated
ingredient are incorporated into feed formulation.
Salmonella may be found in feeds and feed
ingredients which are processed in an unhygienic

environment.
The bacteria may be found in raw fish and fishery
products. Thorough hand washing after contact with

animals is recommended to prevent salmonella
transmission. Contaminated water is also a possible
source of salmonella infection.
3.Microbiological evaluation
Code of practice has been issued for the control of
Salmonella in animal feeds and feed ingredients.

The bacteria in processed food may be damaged as a
result of the dehydration

Process employed during its manufacture, and so a
resuscitation step is necessary to ensure the recovery

of contaminating organisms.
The sample should be tested on the day of receipt or on the 1st
working day that allows the method to be completed if the test is

not begun on the day of receipt.
The sample must be stored in a refrigerator until required.

Refrigerated samples should be left at room temperature for at
least 4 h before examination.
The sample should be tested in duplicate 25 g portions for
Salmonella, five 10 g portions for Enterobacteriaceae, and for

rendered material derived from high-risk material duplicate 10 g
portions for Clostridium perfringens
Example of Contaminated Experimental feed
Microbiological Parameter
Control Feed
Experimental Feed
1. Standard plate count (cfu/g)
2.0 x 10-3
6.24 x 10 4
2. Total coli form (MPN/g)
110
>240
3. Total Fungi (cfu/g)
4.9 x 102
3.8 x 102
4. E. coli (MPN/g)
Absent
Absent
5. Salmonella/25g
Absent
Absent
6. S. aureus
Absent
Absent
Microbiological analyses in feeds and ingredients
Salmonella
Total plate count
Mould*
Yeast*
Coliform bacteria
Bacillus cereus*
E. Coli
Staphylococcus aureus*
a. Salmonella
Procedures in Salmonella determination:
1. Fish meal
a. Aseptically weigh 25g sample into sterile blending
container.
b. Add 225ml sterile lactose broth and blend mixture for
2 minutes.
c. Aseptically transfer homogenized mixture to sterile
wide-mouth, screw cap jar (500 ml) or other
appropriate container and let stand for 60 min. at room
temperature with jar securely capped. (If mixture is
powder, blending may be omitted)
d. If samples are in powder form, add lactose broth and
mix thoroughly for 60 min. at room temperature.
Microbiological analyses for feeds and ingredients
e. Mix well by swirling and determine pH with test
paper. Adjust pH, if necessary to 6.8.
f. Add up to 2.25 ml steamed (15 min) Tergitol Anionic
7 or Triton X-100 and mix well. (actual quantity needed will
depend on the composition of the raw material).
g. Loosen jar caps turn and incubate sample
mixtures for 24H at 350C.
h. Tighten lid and gently shake incubated sample
Microbiological analyses of feeds and ingredients
i. Transfer 0.1 ml mixture to 10 ml RappaportVassiliadis (RV) medium and another 1 ml mixture to

10 ml tetrathionate (TT) broth.
j. Incubate RV medium for 24H at 420C (for high
microbial load) and and TT broth for 24 H at 430C.
K. Mix and streak 3 mm loopful (10 l) incubated TT
broth on bismuth sulfite BS agar. Prepare BS plates
the day before streaking and store in dark at room
temperature until streaked.
Staphylococcus aureus is a gram positive coccal
bacterium, that is a member of the Firmicutes, and is

frequently found in the human respiratory tract and
on the skin.
It is positive for catalase and nitrate reduction.
S. aureus is not always pathogenic, but it is a common
cause of food poisoning.
S. aureus can survive from hours to weeks, or even
months, on dry environmental surfaces, depending on
strain.
a. Staphylococcus aureus
Procedures in S. aureus determination:
1.
2.
3.
4.
5.
aseptically transfer 1 ml sample suspension to 3 plates of Baird-Parker

agar, distributing 1 ml of inoculum equitably to 3 plates.
Spread inoculum over surface of agar plate, using sterile bent glass
streaking rod.
Select plates containing 20-200 colonies.
Count and record colonies.
Report this number as number of S. aureus/g
of food tested.
Plate Count Agar (PCA), also called Standard
Methods Agar (SMA), is a microbiological growth

medium commonly used to assess or to monitor "total"
or viable bacterial growth of a sample. PCA is not a
selective medium.
The composition of plate count agar may vary, but
typically it contains the following: 0.5% peptone; one
0.25% yeast extract; 0.1% glucose; 1.5% agar; pH
adjusted to neutral at 25oC.
Three methods of how to calculate the amount of bacteria in a plate.
The dilution table is as follows:
Unknown
Volume Transferred
Diluent Volume Total Volume
Dilution A
1 ml
999
1000
Dilution B
1 ml
99
100
Dilution C
1 ml
9
10
100 l of Dilution D is used to inoculate two nutrient agar plates. After
incubation the plates show 30 and 31 colonies of bacteria. The average
number of bacteria colonies is 31.
METHOD 1.
Express the dilution and the inoculation in scientific format. (This
value is found by dividing the Volume Transferred by the Total
Volume.)
Dilution A
1x10-3
Dilution B
1x10-2
Dilution C
1x10-1
Inoculation
1x10-1
Calculate the amount of bacteria in the original solution by:
31/(10-3 x 10-2 x 10-1 x 10-1) = 3x108 = no. of bacteria in the
original solution.
The plate count simulation coefficient is always expressed as a
whole number. The answer is 3x108 not 3.1x108.
Method 2
Calculate the ratios of the Total Volume / Volume

Transferred and then multiply it by the colonies counted
on the plate.
1,000 (Dilution A) =
1000 / 1
100 (Dilution B) =
100 / 1
10 (Dilution C) =
10 / 1
10 (inoculation of 100 or 0.1ml) = 10
31 (colonies counted on plate)
300,000,000 = 3x108 = no. of bacteria in the original solution
The plate count simulation coefficient is always expressed
as a whole number. The answer is 3x108 not 3.1x108.
Method 3
To use this method three criteria must be met:

1.the number of bacteria colonies on the plate must be
between 10 and 90
2. all dilutions must be done as 1 to 9, 1 to 99 or 1 to 999
3. inoculation selected must be 100
The coefficient for this solution will be 3. Use the 10's digit from
the plate count.
The exponent for this problem can be determined by counting
zeros.
Count all the zeros for Dilution A, B, and C and then add 2. The
exponent for this problem is 8 (6 zeros in the dilutions +2).
The total number of bacteria in the original sample is 3x108.
To compute for colony plate counts???
Problem:
a small volume e.g. 0.1ml from one of the tubes is transferred

onto an agar plate, where it is spread out over the surface.
Since each colony comes from a single starting bacterium, the
number of colonies = the number of (living) bacteria in the 0.1ml
sample that was spread on the plate. By working backwards it is
possible to calculate how many bacteria there were in the
original 1ml of overnight culture used to make the dilution
series.
The three plates illustrated below are the results of incubating agar plates
overnight after applying 0.1ml from one of the tubes in a bacterial dilution
series. Each plate used a different starting culture.
Using the colony counts and dilution factors given, work out what the original
overnight culture densities were (in bacteria/ml).
Plate A - Diluted 10-fold three times.

80 colonies
Plate B - Diluted 10-fold five times.
46 colonies
Plate C - Diluted 10-fold four times.
147 colonies.
Answer
A: 80 cfu/0.1 ml x 10 x 10 x 10 = 800,000 cfu/ml or 0.8 x
10^6
B: 46/0.1 ml x 10^5 = 46,000,000 cfu/ml or 46 x 10^6
C: 147 cfu/0.1 ml x 10^4 = 14,700,000 cfu/ml or 14.7 x
10^6
Note: cfu means colony forming unit =a single-celled
bacterium.
Series of serial dilutions
Procedure for inoculating a nutrient

agar slant from an agar plate
Colony Counter
Example of Contaminated Experimental feed
Microbiological Parameter
Control Feed
Experimental Feed
1. Standard plate count (cfu/g)
2.0 x 10-3
6.24 x 10 4
2. Total coli form (MPN/g)
110
>240
3. Total Fungi (cfu/g)
4.9 x 102
3.8 x 102
4. E. coli (MPN/g)
Absent
Absent
5. Salmonella/25g
Absent
Absent
6. S. aureus
Absent
Absent
4. Biological Evaluation
does not provide any information about chemical composition
but offers a more accurate estimate of nutritional value and the
efficiency to produce growth and maintain a healthy organism.
live organisms are utilized to conduct well-designed feeding
trials to evaluate the specific effect of a particular nutrient or feed
formulation.
provides information that ascertain the true value of the
feedstuff to the organism.
more preferred method and the ultimate test of performance.

Disadvantages of being time consuming, expensive to conduct and
requires specialized facilities for holding live animal.
1. Feeding Experiment
feeding trials are conducted to determine the performance
of a complete diet or a particular ingredient of interest.
is usually done in tanks, in ponds, or in cages using fish as
test animals. In a laboratory experiment, environmental
conditions are easily kept constant.
measure of a performance such as growth, survival, feed
utilization is used to evaluate the adequacy of an ingredient or
the feed.
more a specific measures of performance such as the
retention or loss of a specific nutrient in the body of the
organism, shifts in enzyme activity or the ability of the
organism to survive a specific environmental challenge. (e.g.
shifts in temperature and salinity and exposure to pathogenic
organisms are needed.
feeding trials should be conducted under strict experimental
conditions, which include environmental monitoring, adequate
replication, and the manipulation of only one or a few
variables at a time.
Parameters to be monitored in a feeding
experiment:
Growth
Efficiency of feed utilization
Digestibility of nutrients
Efficiency of protein utilization
Survival rate
Body composition of fish samples
Biological parameters
Histological changes in tissues
Clinical signs
Reproductive performance
Two most common measures of response to a particular ingredient or
feed are the following:
1. GROWTH
- measured as function of weight, length, or specific nutrient
gain (e.g. protein).
- increases in weight gain through muscle growth and
deposition of specific
biochemical components such as
proteins or lipids.
- weight gain caused by excessive deposition of lipid in the
adipose tissue is undesirable because it decreases yield and
may adversely influence shelf life, resulting in human health
concerns.
- when a diet is evaluated by means of a growth trial, the
performance parameter measured, e.g. growth as weight gain
should be complemented by an analysis of the proximate
composition of the carcass of the organism prior to and
following feed administration.
2. FEED UTILIZATION
- describes to what extent food eaten by the organism is actually
converted into growth.
- particularly critical when comparing the economic cost of feeds
and their potential for polluting the culture environment.
- since the method of feeding will influence the degree of feed
utilization, the type of feeding strategy should be welldocumented in terms of ration size (restricted versus excess) as
well as the number of feedings per day.
In carrying out a feeding experiment, the following factors
have to be considered:
a. the objective of the study must be clearly defined
b. experimental treatments and statistical design
appropriate to the objective of the experiment must
be carefully selected.
c. the experimental fish species must be clearly identified

d. experimental tanks should be large enough to allow
substantial fish growth.
e. duration of feeding experiment should be long enough
at least 8 weeks, to allow fish to manifest definite growth

trends and significant differences in response parameters
as affected by dietary treatments.
f. It is best to know the data to be gathered, sampling
frequency, number of samples per replicate, and statistical
methods to be used in the data analysis beforehand.
Laboratory set-up for biological

evaluation of aquatic feeds
Length of time required to conduct a feeding trial is
dependent on the following factors:

Objectives of the experiment
Species utilized
Age of the animal
Growth trial should be of sufficient duration to
produce relatively large increases in growth and

statistically significant differences between dietary
treatments. (e.g. 14-28 days for larvae, 6-8 weeks for
juveniles, 14-18 weeks for larger fish.
Although purified diets produce slower growth,
growth rates in the laboratory should be comparable to

those achieved under natural conditions. (e.g. ponds)
where both high quality feeds and natural productivity
are available as food sources.
Ingredients for making the experimental diets should
be well characterized so that composition is defined.
The final feed product should of suitable size and
texture for the animals to consume easily.
Animals should be fed according to a well defined
feeding ration which must be adjusted as the animal
grows.
Types of feeding include the following:
1. Restrictive ration- where feed is offered based on a
fixed rate of the animal body weight below satiation.
2. In excess- where feed is offered in a fixed rate that is in
excess of what the animals can consume.
3. Apparent satiation- where food is offered during a
specific period of time until the test animal stop
consuming feed.
o Generally food should be offered to a semi-continous
basis to larvae 4x a day for small juveniles, 2x daily for

large juveniles and once daily to sub-adults.
Other parameters to be monitored in a feeding
experiment:
1. Digestibility
-a measure of biological availability of nutrients or energy in
the ingredient whereas absorption refers to actual uptake .
- methods commonly used for digestibility are the following:
a. Apparent digestibility
-the most direct method to estimate digestibility
which involves feeding a specific amount of an experimental
diet and recording the quantity of feed consumed and feces
produced.
- amount of a particular nutrient in the feces is
subtracted from the initial quantity in the test feed, the
difference represents the amount of nutrient absorbed by the
animal
Techniques used to approximate commercial
conditions.
Tank studies-where outdoor tanks in which natural
productivity is allowed to become established are used
as replicates.
2. Cage studies where cages are either floated in the
pond or a fixed in the pond bottom are used as
replicates.
3. Pond studies where small ponds are used as
replicates.
1.
Feed Digestibility Set-up
Apparent Digestibility (AD)

- means that the feces also contain endogenous fecal
excretion in addition to unabsorbed feed hence the
digestibility estimate can be an underestimate because
some of the nutrient present in the feces could come from
endogenously produced waste
b. True Digestibility
- estimates of endogenous fecal excretions can be obtained
by feeding a diet that does not contain the nutrient being
tested and then determining the amount of nutrient in the
feces.
Apparent digestibility coefficients of ingredients for rainbow

trout (Cho et al, 1982; Cho and Kaushik,1990; Bureau et al.,
1990.
Apparent digestibility coefficients (%)
Ingredients
Dry Matter
Crude protein
Blood meal (spray-dried)
91
96
Blood meal (flame-dried)
55
16
Corn gluten meal
80
96
Fish meal (herring)
85
92
Meat and bone meal
70
85
Soybean meal full-fat,cooked
78
89
Soybean meal,dehulled
74
89
Manufacturing characteristics and apparent digestibility

coefficients of dry matter (DM) and CP of rendered
animal protein ingredients from various origins (Bureau and
Hua, 2007)
Ingredients
Processing conditions
ADC
DM
CP
Feather meal
1
steam hydrolysis,30 min @276kPa, disc dryer
82
81
steam hydrolysis,40 min @276kPa, steam tube dryer
84
87
Meat & Bone meal

1
125-1350C,20-30 min,17-34kPa
61
83
1330C,30-40 min,54kPa
72
88
whole blood, spray drier
92
96
steam coagulated blood, rotoplate drier
82
82
Blood meal
c. Inert Markers
- less time consuming method to obtain estimates of
digestibility.
- animals are fed a diet that contains an inert indigestible
marker such as chromic oxide (0.5-1% for several days).
The quantity of the nutrient of interest relative to the inert
marker can be determined in the feed and feces.
-% digestibility is calculated as follows:
Problems and sources of error with digestibility measurements:
1. Digestibility values can vary relative to factors such as the ff.:
a. size and age of the animal
b. type of feed processing and processing conditions,
c. environmental parameters
d. interactions with other ingredients and or nutrients in the
diet
e. method of collecting the feces
f. type of inert marker used
g. leaching of nutrients
( The inert marker method is the easiest and best to determine the apparent
digestibility of an ingredient and its associated nutrients quickly).
d. Tracer Studies
- radio and stable isotopes are used as tracers to track either ingestion,
digestion and assimilation of dietary nutrients. These isotopes are added
to the diet and utilized as tracer by determining the amount deposited in
the animal tissue.
- difficulties such as the possible loss and recycling of the tracer through
metabolism are associated with these technique. Once the tracer is absorb
it can be utilized for synthesis of new tissue or metabolized and excreted as
waste. Solution to these problem is to use a twin tracer technique to
account for labelled nutrient losses.
e. In vitro Digestibility
- this technique rely on the use of digestive enzyme extracted from the
organisms under study or commercial enzyme.
- enzymes are added to a sample of ingredient being tested and
digestion is measured in vitro by the following methods:
1.
pH-drop method. As proteolytic enzymes attack the peptide bonds

of proteins hydrogen is released and the pH of the protein solution
reduced. The pH reduction is highly and positively correlated with
the degree of protein digestion.
2.
pH-stat method. To keep digestive enzymes close to their optimal

pH, NaOH is added. The amount of NaOH consume is proportional
to the degree of protein hydrolysis and is highly correlated to in vivo
apparent protein digestibility.
Ways of evaluating the availability of specific nutrient in the diet.
1.
Nutrient retention or deposition
2.
Electrical conductivity
-an alternative method based on the different electrical properties of
lipids and water can be used.
- application of an electro-magnetic field and measuring the
different electrical conductivity that can measure the amount of lipid
and water in alive organism.
Method to asses the quality of proteins in the feed
1. Protein efficiency ratio- assess the protein quality by
comparing different protein sources in terms of fish
weight gain per unit of protein fed.
2. Apparent net protein utilization (ANPU)- measurements of
the protein content of the test animal at the start and end of
the experiment, combine with an estimate of the digestibility
value of protein of interest (digestibility coefficient) can be
used to estimate ANPU.
3. True net protein utilization (TNPU) determination is done

by feeding a protein free diet for the same length of time and
determining the change in carcass protein.
4. Biological Value (BV) measures the nutrient excretion
during a period of time. For e.g. All N2 excreted in the
feces, urine, and gills is measured and compared to the
total nitrogen fed.
5. True biological value- an estimate of endogenous loss of

nutrient in question by feeding a N2 free diet.
Alternative measurements to determine nutritional status of
small size organisms (larvae)
1.
RNA/DNA ratio- the quantity of ribonucleic acid (RNA),

the transcriptor and translator of genetic information is
directly proportional to protein synthesis inside the cell.
- the ratio of RNA to DNA correlates
well with growth/protein synthesis and nutritional status
of the fish. High RNA/DNA ratios indicate adequate
growth and nutritional status while low ratios indicate
poor nutritional conditions.
2. Challenge test for the larvae- animals are exposed to
stressful condition such as removing them from

water for a few seconds or exposing them to high or
low salinity. Determination of cumulative mortality
follows.
- weak fish larvae with poor nutritional condition
will not be able to survive this kind of conditions.
Measures of growth and weight gain determine the effects

of aqua feeds on culture species
Growth and weight gain
Efficiency of feed utilization
Digestibility of nutrients
Efficiency of protein utilization
Survival Rate
Quality issues for fats and oils

All marine oils, most plant oils and many animal fats
are rich in polyunsaturated fatty acids (PUFA).

These fat sources are susceptible to rancidity.
Rancid oil must be avoided in the preparation of fish
feeds.
Rancid fat has deleterious effects on nutrients present
in fish feed and health of the fish.
Peroxide value (PV), thiobarbituric acid (TBA) and
anisidine value (AV) are in general parameters in
determine the degree of rancidity in lipid sources.
Acceptable quality parameters for fish oil are as follows:
Quality standards of oils in final feed preparation (Bureau and

Hua, 2007)
Peroxide value
<5meq/kg
Anisidine value
<10
Moisture
<1%
Antioxidant
<500ppm
No vitamin fortification
Clean odor
Summary
The choice of high quality feedstuff for incorporation into aqua
feeds is very crucial to the success of an aquaculture venture.
Proper methods of feed evaluation should be applied to obtain
an effective feed.
Microbiological method of evaluating feed utilizes various
microorganisms in order to determine the nutrient quality of

feeds/feed ingredients.
Biological method of evaluating feed involves actual feeding
experiments and gives a more accurate estimate of feed

utilization.
Types of Feed
1. dry meals/semi-moist pellet/wet diet
10% M.C.
30-45%M.C.
45-70% M.C.
2. crumbles
larval feed
3. hard pellets
for all stages 10% M.C.

4. expanded or floating pellets
feed with lighter density

5. flake diet
feed with lighter density

96

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Myrna B. Teruel, Ph.

systematic evaluation of feedstuffs and feed using

Feed and ingredient evaluations

ingredients are analyzed by a series of physical , chemical,

feed evaluation. Hence, a variety of evaluation/assessment

Evaluation of Feedstuffs and Aquafeeds

Methods of Feed/Ingredient Evaluation

Fast, cheap, easy

High development cost

utilizes various microorganisms (e.g. Lactobacillus

amino acid determination

isomer and no activity for the d-isomer.

Hydrolyze samples by autoclaving with 2N HCl at 15 Ibs.

pressure for 10 hours for maximum liberation of amino acids.

Express results as per cent of each amino acid in the sample as

analyzed and as per cent in the crude protein (calculated to 16%

3 purified proteins (casein, egg albumin and gelatin) are usually

analysis and calculated from the values obtained for the

No.1 (determined) 2.49

The amount of each amino acid contributed by the various

Microbiological Procedure for

amino acids in absence of appreciable amount of

Stock Solutions for Basal Media

For detailed method for the preparation of stock solutions, refer to

Stock cultures of test organisms

For use in assay of

For use in assay of

Incubate (16-24 h, 34C)

For the reference standard

assayed) perform assay, determination and

Vitamin B12 (Cobalamine)

Microbiological Method of Feed

comparison of the stimulation of growth of bacteria by

for specific vitamin

Stock Cultures of Test Organisms

*Before using new culture in assay, make several successive transfers of

1. Vitamin B12 (Cobalamin)

containing 0.1 g of Vitamin B12

(2000) 952.20 for the preparation

Vitamin B12 (Cobalamine)

cobalamin, is important as it helps the body to convert

1210C but very unstable in the presence of light.

cyanocobalamine should be done in the dark room.

Vitamin B12 (Cobalamine)

2. Incubate (624 h between 30

5. Wash cells with

Test Solution (AOAC 2000)

Vitamin B12 (Cobalamine)

autoclave at 121123C reaching

add H2O and

Cool and inoculate each

Vitamin B12 (Cobalamine)

2. Niacin and Niacinamide (vitamin B6)

Refer to AOAC Official method (2000) 944.13 for the

preparation of Basal Medium Stock Solution and

Niacin and Niacinamide

3. Autoclave (15 min,

4. Wash cells with 0.9%

4. Cool and store in

Niacin and Niacinamide

preparation of test solution

Proceed to assay using standard solution, test