2-2014 9.03Biological and Microbiological Evaluation of aquafeeds and feedstuffs-MBT.pdf

7/23/2019 2-2014 9.03Biological and Microbiological Evaluation of aquafeeds and feedstuffs-MBT.pdf

http://slidepdf.com/reader/full/2-2014-903biological-and-microbiological-evaluation-of-aquafeeds-and-feedstuffs-mbtpdf 1/96

Myrna B. Teruel, Ph. D

Scientist

Aquaculture DepartmentSoutheast 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 andfinished feeds should, therefore, undergo the process of evaluation

and quality control in order to produce high quality feed.



systematic evaluation of feedstuffs and feed usingphysical, chemical, microbiological, and biological methods

is necessary to assure their effectiveness when fed to fish.

this procedure starts from procurement of feedstuffs andcontinues to feed processing until manufacture and storageof the finished product.

the finished feed must contain all the nutrients requiredby 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 anassessment to ensure that the final product is of highquality and that deleterious effects of the produce are avoided.

No single test will provide the necessary data for adequatefeed evaluation. Hence, a variety of evaluation/assessmentmethods are utilized to provide information required toassess quality and nutritional value of feeds and feedingredients.



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 andfeedstuffs.

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

Evaluation of Feedstuffs and Aquafeeds



Methods of Feed/Ingredient Evaluation Approaches Advantages DisadvantagesIn vivo approaches

On-farm experiment Reliable Costly; Experimental

designs limitations

Growth/digestibility Reliable Costly; limited accessibility

Lab approaches

In vitro assays Fast; reliable High development cost

Proximate analyses Fast, cheap, easy Analyticalerrors/limitations



a method of feed/feed ingredients evaluation thatutilizes various microorganisms (e.g. Lactobacillusrhamnosus; Enterococcus hirae; Pediococcusacidilactica) in order to determine the nutritionalquality of a product.

3. Microbiological Evaluation




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 theamino 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 acidadded. 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 cellspresent. Pure cultures need to be used in this

kind of evaluation.




3. Microbiological Evaluationa. Determining amino acid composition rapid development of microbiological methods for

amino acid determination

has greatly facilitated their application to theestimation of amino acids in feeds and feedingredients.

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 feedsand feed ingredients (e. g. corn, corn gluten meal, meatand bone scraps, soybean oil, soybean meal, alfalfa leafmeal)are finely ground prior to assay to assurehomogeneity.

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 forassay.

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 usuallyused as standards.



3. Microbiological Evaluation Amino acid content of rations determined by actual

analysis and calculated from the values obtained for thecomponents. All values are expressed as per cent in theration.

• Leucine Valine Isoleucine Phenylalanine

No.1 (determined) 2.49 1.17 1.06 1.07No.1 (calculated) 2.54 1.38 1.10 1.14No. 2 (determined) 2.47 1.27 1.10 1.14

No. 2 (calculated) 2.64 1.42 1.21 1.19

The amount of each amino acid contributed by the variousprotein ingredients was calculated on the basis of thepercentage of the ration in the ingredient.



Applicable only to materials containing free forms ofamino acids in absence of appreciable amount ofprotein

Microbiological Procedure for

Amino acids Assay



Stock Solutions for Basal Media

Stock Solutions forBasal Media

• Amino acid

solution• Adenin-guanine-

uracil solution

• Salt Solution A

• Salt Solution B

• Vitamin Solution I

• Vitamin Solution II

Basal Medium

• In preparingmedium for givenassay, omitparticular aminoacid to be assayedfrom medium

Culture andsuspension media

• Agar culturemedium

• Liquid culturemedium

• Liquid suspensionmedium

For detailed method for the preparation of stock solutions, refer to AOAC 2000 960.47.



Stock cultures of test organisms

• For use in assay ofisoleucine, leucine,

threonine,tryptophan, valine,

arginine andhistidine

Streptococcus

faecalis

• For use in assay ofisoleucine, leucine,

methionine,phenylalanine,

tryptophan, and valine

Lactobacillus

plantarum

• For use in assay oflysine, methionine,

phenylalanine,tyrosine, cystine

and histidine

Pediococcus

cerevisiae



Inoculum

Transfer of cells from stockculture to 10 ml liquidculture medium

Incubate (16-24 h, 34°C)

Centrifuge and decant

Suspend cells in 10 mlsuspension medium

For the reference standardsolutions of amino acids,calibration of photometer

and preparation of testsamples refer to AOAC

2000 960.47.



Assay Using working standard solutions, test solutions,

and basal medium (omitting amino acid being

assayed) perform assay, determination andcalculation as described in AOAC (2000) 960.47.



Microbiological Method ofFeed Evaluation

Vitamin Composition:

Vitamin B12 (Cobalamine)

Niacin (vitamin B3)(nicotinic acid)

Folic acid (vitamin B9)



The microbial assay of vitamins is based upon thecomparison of the stimulation of growth of bacteria bymeasured concentration of vitamin with that produced by

known concentration of standard preparation of vitaminhaving known activity.

growth of microorganisms is proportional to requirement

for specific vitamin

The microbiological assay of vitamins utilizes variousstrains of microbes.

Microbiological Method of Feed

Evaluation



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 agarculture medium.

Incubate 6-24 h at any selected temperature (30-40°C) held constant to within ±0.5°C.

Store in dark room at 10°C

*Before using new culture in assay, make several successive transfers ofculture in 1-2 week period.



1. Vitamin B12 (Cobalamin)

Applicable to materialscontaining ≥0.1 µg of Vitamin B12activity/g or ml

Refer to AOAC Official method(2000) 952.20 for the preparationof Basal Medium Stock Solution

and Cyanocobalamine StandardSolutions (stock solution,intermediate solution and

working solution).



Vitamin B12 (Cobalamine) A sufficient intake of vitamin B12, also known as

cobalamin, is important as it helps the body to convertfood into glucose, which is used to produce energy.

Cobalamine is stable to processing for short periods at1210C but very unstable in the presence of light.

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




Inoculum

1. TransferLactobacillusleichmanni tosterile tube

containing 10 mlliquid culture

medium

2. Incubate (6-

24 h between 30and 40°C)

3. Centrifugeculture

4. decant

5. Wash cells with0.9% NaCl soln

6. Resuspend cellsin 10 ml sterile0.9% NaCl soln

7. Dilute aliquot

with the same solnto give T equivalentto that for dried cell weight of 0.5- 0.75

mg/tube

Read against

suspensionmedium setat 100% T . cellsuspension so

obtained isinoculum

Test Solution (AOAC 2000)

Must contain ≤0.03 mg Na2S2O5/ml




Assay Tubes

Meticulouslycleanse by suitablemeans followed byheating 1-2 h at250°C

autoclave at 121-123°C reaching

this temperaturein ≤ 10 min.

Incubate between 30and 40°C held constantto within ±0.5°C

To test tubes, add in

duplicate or triplicate0.0 (for uninnoculated

blanks), 0.0 (forinoculated blanks), 1.0,2.0, 3.0, 4.0, and 5.0 ml,

respectively, ofstandard solution) add H2O and

5.0 mlappropriate

basal mediumstock solution

and mix

Cool and inoculate eachtube, except 1 set of

duplicate (or replicate)tubes containing 0.0 ml

standard solution(uninoculated), with 1

drop appropriateinoculum.




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 totitration 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 of8-12 ml.

2. Determine amount of vitamin for each level of test solution by interpolationfrom 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 thepreparation of Basal Medium Stock Solution andNiacin Standard Solutions (stock solution,intermediate solution and working solution).

Niacin is sensitive to light and high temperature.



Niacin and Niacinamide Inoculum

1.Dilute basalmedium stock

solution withaqueous solutioncontaining 0.2 µg

niacin/ml

2. Add 10 ml of dilutedmedium to test tubes

3. Autoclave (15 min,121-123°C)

4. Cool and store indark at 10°C

Liquidculture

medium1. Transfer lactobacillus plantarum to sterile

tube containing 10 mlliquid culture medium

2. Incubate 6-24 h(between 30 and 40 °C

3. Centrifuge cultureand decantsupernatant

4. Wash cells with 0.9%NaCl soln

5. Resuspend cells in0.9% NaCl soln

6. Cell suspension soobtained is inoculum

Inoculum



Niacin and Niacinamide Test Solution

Refer to AOAC Official method (2000) 944.13 for the

preparation of test solution

Proceed to assay using standard solution, testsolution, basal medium stock solution and inoculum.



Niacin and Niacinamide

Assay Tubes









5.0 mlappropriate


and mix







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 measurepotentiometrically.

* Disregard results of assay if response at inoculated blank level isequivalent 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 totitration of 8-12 ml.



Niacin (Vitamin B3)

2. Determine amount of vitamin for each level of testsolution 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 asniacinamide.



3. Folic acid (Vitamin B9)

Applicable only to materials containing free forms offolic acid.

Refer to AOAC Official method (2000) 944.12 for thepreparation of Basal Medium Stock Solution and Folicacid 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 liquidculture medium

• Incubate 6-24 h (between 30 and 40°C)

• 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 testsolution.

Perform assay using standard solution, test solution, basal medium stocksolution and inoculum.



Folic Acid (Vitamin B9)

Assay Tubes









5.0 mlappropriate


and mix







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 totitration 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 of8-12 ml.

2. Determine amount of vitamin for each level of test solution by interpolationfrom 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 areinsufficient for calculating potency of product.

If number of acceptable values remaining is ≥2/3 of originalnumber of tubes, calculate potency of product from average ofthem.



3. Microbiological evaluation Animal feeds

Aquatic animals reared intensively require large amounts of plant oranimal protein in the feed. This material is prepared from meat,

offal, bones, blood or feathers, or combinations. Animalproteins often contain high level of Salmonella which dependson the initial contamination of the raw materials and on thehygiene 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 duringprocessing and feed preparation (inadequate cooking andstorage procedures).



3. Microbiological Evaluation What is salmonella?

Salmonella is bacteria that can cause a gastrointestinalinfection known as salmonellosis. Usuallysalmonellosis is referred to as "salmonella." Thisinfection can occur in humans and animals.

People become infected with salmonella by swallowing

the bacterium. This can happen when contaminatedingredient are incorporated into feed formulation.



3. Microbiological Evaluation Salmonella may be found in feeds and feed

ingredients which are processed in an unhygienicenvironment.

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

animals is recommended to prevent salmonellatransmission. Contaminated water is also a possiblesource of salmonella infection.



3.Microbiological evaluationCode 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 aresult of the dehydration

Process employed during its manufacture, and so aresuscitation step is necessary to ensure the recoveryof contaminating organisms.



3. Microbiological evaluation

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 isnot 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 atleast 4 h before examination.

The sample should be tested in duplicate 25 g portions forSalmonella, five 10 g portions for Enterobacteriaceae, and forrendered material derived from high-risk material duplicate 10 gportions for Clostridium perfringens



Microbiological Parameter Control Feed Experimental Feed

1. Standard plate count (cfu/g) 2.0 x 10-3 6.24 x 10 42. 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 Absent6. S. aureus Absent Absent

3.Microbiological evaluationExample of Contaminated Experimental feed



Microbiological analyses in feeds and ingredients

•

Salmonella

•Total plate count

•Mould*

•Yeast*

•Coliform bacteria

•Bacillus cereus*

•E. Coli

•Staphylococcus aureus*

3.Microbiological evaluation



a. Salmonella

Procedures in Salmonella determination:

1. Fish meala. Aseptically weigh 25g sample into sterile blendingcontainer.

b. Add 225ml sterile lactose broth and blend mixture for

2 minutes.





3. Microbiological evaluation

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 andmix thoroughly for 60 min. at room temperature.




3. Microbiological evaluation Microbiological analyses for feeds and ingredients

e. Mix well by swirling and determine pH with testpaper. Adjust pH, if necessary to 6.8.

f. Add up to 2.25 ml steamed (15 min) Tergitol Anionic7 or Triton X-100 and mix well. (actual quantity needed willdepend on the composition of the raw material).

g. Loosen jar caps ¼ turn and incubate samplemixtures for 24H at 350C.

h. Tighten lid and gently shake incubated sample



3. Microbiological Evaluation Microbiological analyses of feeds and ingredients

i. Transfer 0.1 ml mixture to 10 ml Rappaport- Vassiliadis (RV) medium and another 1 ml mixture to10 ml tetrathionate (TT) broth.

j. Incubate RV medium for 24H at 420C (for high

microbial load) and and TT broth for 24 H at 430

C.K. Mix and streak 3 mm loopful (10 µl) incubated TTbroth on bismuth sulfite BS agar. Prepare BS platesthe day before streaking and store in dark at roomtemperature until streaked.



Staphylococcus aureus is a gram positive coccalbacterium, that is a member of the Firmicutes, and isfrequently 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 commoncause of food poisoning.

S. aureus can survive from hours to weeks, or evenmonths, on dry environmental surfaces, depending onstrain.




a. Staphylococcus aureus

Procedures in S. aureus determination:

1. aseptically transfer 1 ml sample suspension to 3 plates of Baird-Parkeragar, distributing 1 ml of inoculum equitably to 3 plates.

2. Spread inoculum over surface of agar plate, using sterile bent glassstreaking rod.

3. Select plates containing 20-200 colonies.

4. Count and record colonies.

5. Report this number as number of S. aureus/g

of food tested.




3. Microbiological Evaluation Plate Count Agar (PCA ), also called Standard

Methods Agar (SMA), is a microbiological growthmedium commonly used to assess or to monitor "total"

or viable bacterial growth of a sample. PCA is not aselective medium.

The composition of plate count agar may vary, buttypically it contains the following: 0.5% peptone; one

0.25% yeast extract; 0.1% glucose; 1.5% agar; pHadjusted to neutral at 25oC.

http://en.wikipedia.org/wiki/Peptone

http://en.wikipedia.org/wiki/Peptone



3. Microbiological Evaluation 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 µl of Dilution D is used to inoculate two nutrient agar plates. After

incubation the plates show 30 and 31 colonies of bacteria. The averagenumber of bacteria colonies is 31.



3. Microbiological Evaluation 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.



3. Microbiological Evaluation Method 2 Calculate the ratios of the Total Volume / VolumeTransferred and then multiply it by the colonies countedon the plate. 1,000 (Dilution A) = 1000 / 1

100 (Dilution B) = 100 / 110 (Dilution C) = 10 / 110 (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 expressedas a whole number. The answer is 3x108 not 3.1x108.



3. Microbiological Evaluation 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.



3. Microbiological Evaluation To compute for colony plate counts???

Problem:

a small volume e.g. 0.1ml from one of the tubes is transferredonto an agar plate, where it is spread out over the surface.

Since each colony comes from a single starting bacterium, thenumber of colonies = the number of (living) bacteria in the 0.1mlsample that was spread on the plate. By working backwards it is

possible to calculate how many bacteria there were in theoriginal 1ml of overnight culture used to make the dilutionseries.



The three plates illustrated below are the results of incubating agar platesovernight after applying 0.1ml from one of the tubes in a bacterial dilutionseries. Each plate used a different starting culture.

Using the colony counts and dilution factors given, work out what the originalovernight 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 x10^6B: 46/0.1 ml x 10^5 = 46,000,000 cfu/ml or 46 x 10^6C: 147 cfu/0.1 ml x 10^4 = 14,700,000 cfu/ml or 14.7 x10^6

Note: cfu means colony forming unit =a single-celledbacterium.




Series of serial dilutions

Procedure for inoculating a nutrientagar slant from an agar plate



Colony Counter



Microbiological Parameter Control Feed Experimental Feed

1. Standard plate count (cfu/g) 2.0 x 10-3 6.24 x 10 42. 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

3.Microbiological evaluationExample of Contaminated Experimental feed



4. Biological Evaluation

does not provide any information about chemical compositionbut offers a more accurate estimate of nutritional value and theefficiency to produce growth and maintain a healthy organism.

live organisms are utilized to conduct well-designed feedingtrials to evaluate the specific effect of a particular nutrient or feedformulation.

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 andrequires 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, feedutilization 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 theorganism, 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


4 Biological Evaluation




Two most common measures of response to a particular ingredient orfeed are the following:

1. GROWTH- measured as function of weight, length, or specific nutrientgain (e.g. protein).- increases in weight gain through muscle growth and

deposition of specific biochemical components such asproteins or lipids.- weight gain caused by excessive deposition of lipid in theadipose tissue is undesirable because it decreases yield andmay adversely influence shelf life, resulting in human healthconcerns.- when a diet is evaluated by means of a growth trial, theperformance parameter measured, e.g. growth as weight gainshould be complemented by an analysis of the proximatecomposition of the carcass of the organism prior to andfollowing feed administration.



2. FEED UTILIZATION- describes to what extent food eaten by the organism is actuallyconverted into growth.

- particularly critical when comparing the economic cost of feedsand their potential for polluting the culture environment.

- since the method of feeding will influence the degree of feedutilization, the type of feeding strategy should be well-

documented 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 factorshave 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 isdependent 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 andstatistically significant differences between dietarytreatments. (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 tothose achieved under natural conditions. (e.g. ponds) where both high quality feeds and natural productivityare available as food sources.

Ingredients for making the experimental diets shouldbe well characterized so that composition is defined.

The final feed product should of suitable size andtexture for the animals to consume easily.

Animals should be fed according to a well definedfeeding ration which must be adjusted as the animalgrows.




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 inexcess of what the animals can consume.

3. Apparent satiation- where food is offered during aspecific period of time until the test animal stopconsuming feed.

o Generally food should be offered to a semi-continousbasis to larvae 4x a day for small juveniles, 2x daily forlarge juveniles and once daily to sub-adults.




Other parameters to be monitored in a feedingexperiment:

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 commercialconditions.

1. Tank studies-where outdoor tanks in which naturalproductivity 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 asreplicates.

3. Pond studies – where small ponds are used asreplicates.




Feed Digestibility Set-up



Apparent Digestibility (AD)- means that the feces also contain endogenous fecalexcretion in addition to unabsorbed feed hence thedigestibility estimate can be an underestimate because

some of the nutrient present in the feces could come fromendogenously produced waste




b. True Digestibility

- estimates of endogenous fecal excretions can be obtainedby feeding a diet that does not contain the nutrient being

tested and then determining the amount of nutrient in thefeces.




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 andHua, 2007)

Ingredients Processing conditions ADC

DM CP

Feather meal1 steam hydrolysis,30 min @276kPa, disc dryer 82 81

2 steam hydrolysis,40 min @276kPa, steam tube dryer 84 87

Meat & Bone meal

1 125-1350C,20-30 min,17-34kPa 61 83

2 1330C,30-40 min,54kPa 72 88

Blood meal

1 whole blood, spray drier 92 96

2 steam coagulated blood, rotoplate drier 82 82



c. Inert Markers- less time consuming method to obtain estimates ofdigestibility.- animals are fed a diet that contains an inert indigestiblemarker such as chromic oxide (0.5-1% for several days).The quantity of the nutrient of interest relative to the inertmarker 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 thediet

e. method of collecting the feces

f. type of inert marker usedg. leaching of nutrients

( The inert marker method is the easiest and best to determine the apparentdigestibility 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 addedto the diet and utilized as tracer by determining the amount deposited inthe animal tissue.

- difficulties such as the possible loss and recycling of the tracer throughmetabolism are associated with these technique. Once the tracer is absorbit 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 toaccount for labelled nutrient losses.

e. In vitro Digestibility

- this technique rely on the use of digestive enzyme extracted from theorganisms under study or commercial enzyme.




- enzymes are added to a sample of ingredient being tested anddigestion is measured in vitro by the following methods:

1. pH-drop method. As proteolytic enzymes attack the peptide bondsof proteins hydrogen is released and the pH of the protein solutionreduced. The pH reduction is highly and positively correlated withthe degree of protein digestion.

2. pH-stat method. To keep digestive enzymes close to their optimalpH, NaOH is added. The amount of NaOH consume is proportionalto the degree of protein hydrolysis and is highly correlated to in vivoapparent 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 thedifferent electrical conductivity that can measure the amount of lipidand water in alive organism.




Method to asses the quality of proteins in the feed

1. Protein efficiency ratio- assess the protein quality bycomparing different protein sources in terms of fish

weight gain per unit of protein fed.




2. Apparent net protein utilization (ANPU)- measurements ofthe protein content of the test animal at the start and end ofthe experiment, combine with an estimate of the digestibility value of protein of interest (digestibility coefficient) can beused to estimate ANPU.

3. True net protein utilization (TNPU) – determination is doneby 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 excretionduring a period of time. For e.g. All N2 excreted in thefeces, 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 ofsmall size organisms (larvae)

1. RNA/DNA ratio- the quantity of ribonucleic acid (RNA),the transcriptor and translator of genetic information isdirectly proportional to protein synthesis inside the cell.

- the ratio of RNA to DNA correlates

well with growth/protein synthesis and nutritional statusof the fish. High RNA/DNA ratios indicate adequategrowth and nutritional status while low ratios indicatepoor nutritional conditions.




2. Challenge test for the larvae- animals are exposed tostressful condition such as removing them from

water for a few seconds or exposing them to high orlow salinity. Determination of cumulative mortalityfollows.

- weak fish larvae with poor nutritional condition

will not be able to survive this kind of conditions.




Growth and weight gain

Measures of growth and weight gain determine the effects

of aqua feeds on culture species



Efficiency of feed utilization

Digestibility of nutrients

Effi i f t i tili ti



Efficiency of protein utilization

Survival Rate



Quality issues for fats and oils All marine oils, most plant oils and many animal fatsare rich in polyunsaturated fatty acids (PUFA).

These fat sources are susceptible to rancidity.

Rancid oil must be avoided in the preparation of fishfeeds.

Rancid fat has deleterious effects on nutrients presentin fish feed and health of the fish.

Peroxide value (PV), thiobarbituric acid (TBA) andanisidine value (AV) are in general parameters indetermine the degree of rancidity in lipid sources.



Quality standards of oils in final feed preparation (Bureau and

Hua, 2007)

Acceptable quality parameters for fish oil are as follows:

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 variousmicroorganisms in order to determine the nutrient quality offeeds/feed ingredients.

Biological method of evaluating feed involves actual feedingexperiments and gives a more accurate estimate of feedutilization.



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

Documents

2-2014 9.03Biological and Microbiological Evaluation of aquafeeds and feedstuffs-MBT.pdf