Dr. Waqas Nawaz PMAS Arid Agriculture University Rawalpindi, Pkaistan

1. Special tests for
anti-nutritional and
toxic factors in
poultry feed
11-ARID-961, 962, 969, 972, 975, 978
Faculty of Veterinary & Animal Sciences
PMAS Arid Agriculture University, Rawalpindi

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 Anti-nutritional & Toxic Factors
 Amino Acid Analysis by Ion-Exchange
Chromatography
 Bleach Test for Tennins in Sorgham
 Rapid Test for Tennnins in Barley
 Mycotoxin Analysis
 Aflatoxin Analysis
 Analysis of Ochratoxin
 Analysis of Zearalenone
 Rapid Mycotoxins test (ELISA)
 Analysis of DON
 Analysis of T-2 Toxin

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“Substances which either by themselves or
through their metabolic products, interfere
with food utilization and affect the health and
production of animals”
“Components Of Feed which have toxic effects
and deleterious effects on living cells &
tissues are called toxic factors”

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o Tennins (Sorgham) Bind with dietary protein
o Cyclopropenoid Fatty acids=CFA (cottonseed) Albumin
discoloration
o Gossypol (cottonseed) yolk discoloration
o Lectins (Sorgham grains) Bind with CHO
o Phytates (Sorgham grains) Block absorption of
minerals(Ca, Mg, Fe, Zn, P), Lipid,Proteins
o Sponins (Soybean) Bitter, low intake
o Protease inhibitors (Soybean) Amino Acids available
o DON or Deoxynevalenol or Vomitoxin (Barley) Immunity
o T-2 Toxin (Corn, Oat) Lesions on beak, egg production
o Cyanogens (Linseed, Sorgham) HCN poisoning

5. Reagents (Hydrolyasate):
 6N HCL: 50 ml of concentrated hydrochloric acid added to 50
ml of double distilled water.
 Nor-leucine standard, 25 mole/ml
 Sodium citrate buffer, pH=2.2
Apparatus:
Rotary Evaporator40 mesh sieve

6. Grind sample finely (grind to pass a 40 mesh sieve).
Weigh the hydrolysate tube.
Weigh the sample into the hydrolysate tube, so as to
contain about 30-40 mg of protein. This would be
approximately 60-80 mg of soybean meal samples and 300-
350 mg for corn samples when diluting the hydrolysate 100
times.
Add 6 ml of 6N HCL and 0.6 ml of norleucine standard &
Mix well.
Charge the tube with nitrogen gas and place in an oven at
110ᵒC for 20 hours. After the tube cools, filter the contents
through Whattman No.1 filter paper into a drying tube.

7. . Wash hydrolysate tube with double distilled water and collect in
the same drying tube
Dry the filtrate by evaporating with a rotary evaporator under
vacuum, with the water bath temperature at 48ᵒC.
Dry the filtrate to a slightly wet residue & Wash residue with
distilled water and dry again.
Add 20 ml of citrate buffer and mix well.
Take one ml from last step (C-buffer) and
dilute to 5 ml with citrate buffer.
Filter the diluted sample liquid using
0.2 micron Nucleopore membrane filter.
Determine amino acids by injecting sample
into Amino acid analyzer.

8. Scope:
Applicable to whole grain sorghum
Principle:
Sorghum grain is immersed in a sodium hypochlorite solution (bleach)
containing alkali. The solution dissolves away the outer peri-carp
layer of sorghum grain, revealing the presence of a black pigmented
testa layer in the case of tannin sorghums, or its absence in the case of
non-tannin sorghums.
Bleaching reagent
5g sodium hydroxide is dissolved in 100 ml of 3.5% sodium
hypochlorite solution (commercial/Household bleach). Reagent can be
stored at room temperature in light-proof bottle for up to one month.

9.  Apparatus
Glass beakers (50 ml)
Tea strainer
Aluminum foil
Paper towel

10. One hundred whole, sound sorghum grains are placed in a beaker
Bleaching reagent is added to just cover the sorghum grains
Close beaker with aluminum foil. Too much bleaching reagent will
cause over bleaching and give false negative results. If in doubt repeat
using less reagent.
Incubate beaker at room temperature (20-30 C)for 20
minutes, swirling contents of beaker every 5 minutes
Empty contents of beaker into tea strainer, discarding bleaching
reagent.
Rinse sorghum grains in tea strainer with tap water
Empty contents of tea strainer onto sheet of paper towel.
Spread grains out into a single layer and gentle blot them dry with
another piece of paper towel

11. 1.Take about of 10 gms of sample (Barley) in bottle
2. Add potassium hydroxide and sodium hypochloride solution.
3. Close the bottle and shake till crystals dissolve.
4. Wait for about 15 minutes
Tannin sorghum grains are those grains that are black over the entire
surface of the grain, with the exception of the where the germ is which
is somewhat lighter in colour. Non-tannin sorghum grains are those
which are either completely white or are brown over part of the
surface of the grain

12. 1
• High Performance Liquid Chromatography HPLC
2
• Immuno Assay
3
• Mini-column Method
4
• High Performance Thin Layer Chromatography HPTLC
5
• Spactrophotometry
6
• Thin Layer Chromatography , TLC

13. 1-Sampling
Collect at least 100 subsamples from the whole lot. For eg. from
a truck of 100 bags of maize, collect 100 g maize from each bag
to obtain a total sample size of 10 kg.
Get about 50 - 100 g subsample from the whole sample
employing either coning and quartering method
2-Toxin extraction (using organic solvents i.e. Acetic acid)
3-Clean-up (To remove fat, impurities etc.)
4-Identification & Quantification (TLC, HPLC, ELISA etc.)

14. Principle:
It is the cheapest and most commonly used method. It makes use
of heterogenous equilibrium established during the flow of a
solvent(mobile phase) through a fixed phase (stationary phase)
to separate ≥2 components from materials carried by solvent
(differential migration).
Apparatus:
 Chromatogram
TLC plate
Micropippete/Microcap
TLC Scanner

15. UV Cabnet
TLC Scanner
Micropipette
Chromatogram

16. Spotting the extract:
o Place 5 - 20 µl of sample as a small circular spot (< 5 mm), 1 - 2
cm from the end of the TLC plate. Micropipette may be used for
the purpose. Leave at least 1 cm gap between two adjacent spots.
Developing the plate:
o Place about 50 - 100 ml of mobile phase (solvent) in a tank
o Keep the plate at a slight angle with the spots little above the
upper level of the solvent. Due to capillary action, solvent moves
upward on the plate.
o Allow the solvent to travel at least about 8-10 cms

17.  Detection:
o Air dry the developed plate and view in a UV cabinet under
either longwave (365 nm) or short wave (254 nm) range to
identify the fluorescing mycotoxins. In case of mycotoxins
which do not fluoresce, spray the plate with suitable reagent (like
50 % aqueous H2SO4 , Triflouro,etc). to develop fluorescence.
 Resolving front value (Rf):
o Each mycotoxin has its characteristic color of fluorescence under
UV light and a constant Rf value in a particular developing
solvent(See Table ).
o Rf value is computed using the formula;

18.  Confirmation:
o The presence of mycotoxin can be confirmed either by spraying
the plate with suitable reagents (like 50 % aqueous H2SO4
, Triflouro, Acetic Acid etc).
 Detection by Scanner:
o The fluorescence intensity of sample and standard spots can be
measured by using TLC Scanner / fluorodensitometer to avoid
possible human errors in comparison

20. This is an extension of TLC method. The sample spots on the
developed TLC plate are scraped out along with the sorbent
(silica gel) and extracted with methanol for 3 minutes. The
extract is filtered and the absorbance of the filtrate is measured
in a spectrophotometer at 363 nm.
This is an improvised version of TLC, where sample application
and detection of fluorescence intensity are fully automated and
carried out by using automated sample applicator (like Linomat-
IV of Camag, Switzerland) and densitometer, respectively.
Mycotoxin levels less than 0.1 ppb can be detected by this
method.

21. Conical Flask
100ml Beaker
Spectrophotometer Linomat IVBlender

22.  Reagents:
o 0.2 M NaOH (dissolve 8 g NaOH in water and make up volume
to 1 lit)
o 0.41 M Ferric Chloride (dissolve 66.5 g anhydrous FeCl3 in
water and make up volume to 1 lit)
o 0.03 % H2SO4 (0.3 ml conc. H2SO4+ 999.7 m water)
o Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl in
water and make up volume to 1lit)
Solvents:
o Acetone
o Chloroform
o Developing solvent==Chloroform : Acetone : Water (88 : 12 : 1)

23. Standard:
o Aflatoxin B1 1 µg/ml in Benzene : Acetonitrile (98:2)
Procedure:
o Take 25 g sample in a conical flask, add 100 ml distilled water
and blend for 2 minutes
o Add 150 ml acetone and blend again for 2 minutes
o Filter through Whattman no.1 filter paper and transfer 75 ml of
filtrate to a conical flask containing 3 g cupric carbonate
o Prepare ferric gel by adding 85 ml of 0.2 M NaOH to 15 ml of
0.41 M FeCl3
o Add this mixture to the flask containing extract and cupric
carbonate

24. Mix the contents slowly by swirling movements
Filter through Whattman no. 1 filter paper
Take 100 ml of filtrate in a 250 ml separating funnel
Add 100 ml of 0.03 % H2SO4 and 10 ml of chloroform. Mix the
contents slowly
Collect the chloroform layer into a 100 ml beaker
Add again 10 ml of chloroform to the separating funnel and
repeat the above step. Combine both the chloroform extracts
Take 100 ml potassium wash solution in a separate separating
funnel
Add the chloroform extract to the second separating funnel and
mix it slowly

25. Collect the chloroform layer through anhydrous sodium sulfate
bed drop by drop to remove moisture
Dry the chloroform extract in an oven at 50 C
Dissolve the dried residue in 0.2 ml chloroform and spot on TLC
plat along with the standard
Compare the flourescence intensities of the sample and standard
spots and identify the ones matching with each other
2 ml of final chloroform extract is placed in the column (20 cm
length, 6 mm internal diameter with tapering end 2 mm) and
eluted with chloroform : acetone (9 : 1). Aflatoxin,if present is
trapped as a band above florisil layer which can be viewed under
long wave UV light as a blue fluorescent band

26. Enzyme-Linked Immuno-Sorbant Assay ELISA
RadioImmuno Assay IRA
Immunoaffinity Column Assay ICA

27. Principle :
o Antibody coated column is used to trap the mycotoxin. This
trapped toxin is then eluted using approximate solvent and
quantified in fluorometer.
Equipments :
o Immuno affinity column
o Affinity column stand with syringe
o Cuvette
o Calibrated Fluorometer
o Blender
o Fluted filter paper
Reagent :
oTest developer
o2. Methonol : water
(80 : 20 by volume)
o3. Mycotoxin wash buffer

28. Calibrated Fluorometer
Immuno affinity column
Quartz CuvetteFluted filter paperAffinity column stand with syringe

29.  50 gms of sample + 5 gms of NaCl + 100 ml of methanol water
(80 :20)
Note : NaCl is not added in case of Ochratoxin Test
Blend at high speed and filter
Pipette filtered extract into clean vessel
Aflatoxin, Ochratoxin : 10 ml
Zearalenone : 1 ml
Dilute with purified water and mix
Aflatoxin, Ochratoxin : 40 ml
Zearalenone : 49 ml
Filter
Remove top cap and attach the syringe (cut 1/8 inch bottom of
column )

30. Pass filtered diluted extract at the rate of 1-2 drops/second
Aflatoxin : 2 ml
Ochratoxin, Zearalenone : 10ml
Pass water at the rate of 1-2 drops/second
Aflatoxin, Zearalenone : 5 ml
Ochratoxin : First 10 ml Mycotoxin wash buffer,
Later 10 ml distilled water
Elute toxin in glass cuvette
Aflatoxin, Zearalenone : Pass 1 ml HPLC grade methanol
Ochratoxin : Pass 1.5 ml Ochratoxin eluting solution.
Add 1 ml of developer to the cuvette and mix well
Read in calibrated fluorometer

31. Measured by ELISA
Poultry are the most tolerant of livestock species to
DON. Some studies show that feeding 20-50 ppm
DON has no effect on production.

32. Reagents:
1. Urease enzyme solution
2. Standard Urea Solutions (0, 0.5, 1, 1.5,..........5%)
3. Phenol red indicator (0.1%)
Or
Cresol red indicator (0.1%)
Procedure:
Weigh 10 g. tested sample and add 100 ml of distilled water.
Mix thoroughly and then filter with whatman No. 41 filter paper.
Take 1 ml of tested sample aliquate into white porcelein spot
plate.
Add 2-3 drops of phenol red indicator and then add 2-3 drops of
urease solution.

33. Stand for 3-5 minutes
If Urea presents solution will become red- purple in contrast to
the yellow colour of indicator
Colour can be compared with the colour developed in standard
solution of varying levels of urea.
white porcelein spot plate Whatman #41

34. Reagents :
o 30% ammonium sulphate (dissolve 30 g (NH4)2SO4 in water
and make up volume to 100 ml)
o Celite 545
o Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl in
water and make up volume to 1 lit)
o Sodium sulphate
o Silica gel
o Methanol : H2SO4 ( 1 : 1 v/v )

35. Solvents :
o Methanol : water (1 : 1 v/v)
o Chloroform
o Diethyl ether
o Hexane
o Benzene
o Acetone : Benzene (5 : 95 v/v)
o Developing solvent mixture - Toluene : ethylacetate formic acid
(6 : 3: 1 v/v)
Standard :
o T-2 toxin 50 µg / ml in Benzene or diethyl ether

36. Developing Tank/Chamber
UV cabnet
Pre-coated Silica Gel plate
Horizontal Shaker
Glass Stoppered
Conical Flask
Whatman FP #1

37. Take 50 g of sample in a glass stoppered conical flask
Add 250 ml of methanol : water (1 : 1) and shake for 1 hour
Filter using whatman No.1 filter paper and collect 60 ml of
extract into a beaker
Add 240 ml 30 % (NH4)2SO4 and stir vigorously for 1 minute
Add 20 g of celite and stir for 1 minute
Filter and collect 200 ml of filtrate
Transfer filtrate to a separating funnel
Add 10 ml of chloroform and shake vigorously for 1 minute
Allow the layers to separate and collect the bottom layer into
another separating funnel
Repeat the extraction with another 10 ml of chloroform

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 Combine both the extracts and add 100 ml of
potassium wash solution
 Swirl gently for 30 seconds and let layers
separate
 Drain the lower chloroform layer through a
bed of Sodium sulphate (in a funnel) to dry
and collect 10 ml of clear filtrate
 Column Preparation :
o Plug the bottom of a glass column ( 2x30cm )
with glass wool and add 5 g anhydrous
sodium sulphate. Fill the column to half level
with chloroform and add 10 g silica gel.

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o Wash sides of column with chloroform and
stir to eliminate air bubbles.
o Drain off chloroform leaving about 7 cm
above the upper level of silica gel. Add 15 g
anhydrous sodium sulphate without
disturbing the silica gel.
o Drain off chloroform to the upper level of
sodium sulphate
 Wash the column serially with 50 ml of
diethyl ether and 10 ml of chloroform and
discard the washings

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 Mix 10 ml of sample extract with 30 ml of
hexane and add to the column and slowly
drain until solvent is about 1 cm above
Sodium sulphate
 Add in succession 30 ml benzene and 40 ml
acetone : benzene (5: 95) and discard both
the washings
 Elute T-2 with diethyl ether until 30 ml of
eluate is collected and evaporate the eluate

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 Dissolve the residue in 0.5-1.0 ml diethyl
ether. Spot on TLC along with the standard
(5-20 µL or any other suitable range) and
develop the plate in toluene : ethyl acetate :
formic acid (6 : 3 : 1)
 Air dry the plate and spray with
methanol:H2SO4 (1:1)
 Dry at 110 C for 10 minutes
 Observe blue fluorescence under long wave
UV light (365 nm)
 Compare the intensities of the blue fluorescent spots
of the sample with those of standard and identify the
ones matching each other

42. References:
o Commercial Poultry Nutrition
o Analysis of Toxins by layer chromatography by R.W. Nicol & R.C. Sinha
o Laboratory Manual on Laboratory Control of Animal feed by Dr.
G.Devegowda
o Lecture of Dr. Naeem Tahir (PMAS Arid Agriculture
University, Rawalpindi)
o Simple Sorgham Grain test for tannin by John R.N Taylor
o www.foa.com
o www.hamletprotein.com
o www.allaboutfeed.net
o www.poultryhub.org