Special tests for antinutritional and toxic factors in poultry feeds


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Dr. Waqas Nawaz
PMAS Arid Agriculture University Rawalpindi, Pkaistan

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Special tests for antinutritional and toxic factors in poultry feeds

  1. 1. Special tests foranti-nutritional andtoxic factors inpoultry feed11-ARID-961, 962, 969, 972, 975, 978Faculty of Veterinary & Animal SciencesPMAS Arid Agriculture University, Rawalpindi
  2. 2. More Free PowerPoint Templates at SmileTemplates.com Anti-nutritional & Toxic Factors Amino Acid Analysis by Ion-ExchangeChromatography 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
  3. 3. More Free PowerPoint Templates at SmileTemplates.com“Substances which either by themselves orthrough their metabolic products, interferewith food utilization and affect the health andproduction of animals”“Components Of Feed which have toxic effectsand deleterious effects on living cells &tissues are called toxic factors”
  4. 4. More Free PowerPoint Templates at SmileTemplates.como Tennins (Sorgham) Bind with dietary proteino Cyclopropenoid Fatty acids=CFA (cottonseed) Albumindiscolorationo Gossypol (cottonseed) yolk discolorationo Lectins (Sorgham grains) Bind with CHOo Phytates (Sorgham grains) Block absorption ofminerals(Ca, Mg, Fe, Zn, P), Lipid,Proteinso Sponins (Soybean) Bitter, low intakeo Protease inhibitors (Soybean) Amino Acids availableo DON or Deoxynevalenol or Vomitoxin (Barley) Immunityo T-2 Toxin (Corn, Oat) Lesions on beak, egg productiono Cyanogens (Linseed, Sorgham) HCN poisoning
  5. 5. Reagents (Hydrolyasate): 6N HCL: 50 ml of concentrated hydrochloric acid added to 50ml of double distilled water. Nor-leucine standard, 25 mole/ml Sodium citrate buffer, pH=2.2Apparatus:Rotary Evaporator40 mesh sieve
  6. 6. Grind sample finely (grind to pass a 40 mesh sieve).Weigh the hydrolysate tube.Weigh the sample into the hydrolysate tube, so as tocontain about 30-40 mg of protein. This would beapproximately 60-80 mg of soybean meal samples and 300-350 mg for corn samples when diluting the hydrolysate 100times.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 at110ᵒC for 20 hours. After the tube cools, filter the contentsthrough Whattman No.1 filter paper into a drying tube.
  7. 7. . Wash hydrolysate tube with double distilled water and collect inthe same drying tubeDry the filtrate by evaporating with a rotary evaporator undervacuum, with the water bath temperature at 48ᵒC.Dry the filtrate to a slightly wet residue & Wash residue withdistilled water and dry again.Add 20 ml of citrate buffer and mix well.Take one ml from last step (C-buffer) anddilute to 5 ml with citrate buffer.Filter the diluted sample liquid using0.2 micron Nucleopore membrane filter.Determine amino acids by injecting sampleinto Amino acid analyzer.
  8. 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-carplayer of sorghum grain, revealing the presence of a black pigmentedtesta layer in the case of tannin sorghums, or its absence in the case ofnon-tannin sorghums.Bleaching reagent5g sodium hydroxide is dissolved in 100 ml of 3.5% sodiumhypochlorite solution (commercial/Household bleach). Reagent can bestored at room temperature in light-proof bottle for up to one month.
  9. 9.  ApparatusGlass beakers (50 ml)Tea strainerAluminum foilPaper towel
  10. 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 willcause over bleaching and give false negative results. If in doubt repeatusing less reagent.Incubate beaker at room temperature (20-30 C)for 20minutes, swirling contents of beaker every 5 minutesEmpty contents of beaker into tea strainer, discarding bleachingreagent.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 withanother piece of paper towel
  11. 11. 1.Take about of 10 gms of sample (Barley) in bottle2. Add potassium hydroxide and sodium hypochloride solution.3. Close the bottle and shake till crystals dissolve.4. Wait for about 15 minutesTannin sorghum grains are those grains that are black over the entiresurface of the grain, with the exception of the where the germ is whichis somewhat lighter in colour. Non-tannin sorghum grains are thosewhich are either completely white or are brown over part of thesurface of the grain
  12. 12. 1• High Performance Liquid Chromatography HPLC2• Immuno Assay3• Mini-column Method4• High Performance Thin Layer Chromatography HPTLC5• Spactrophotometry6• Thin Layer Chromatography , TLC
  13. 13. 1-SamplingCollect at least 100 subsamples from the whole lot. For eg. froma truck of 100 bags of maize, collect 100 g maize from each bagto obtain a total sample size of 10 kg.Get about 50 - 100 g subsample from the whole sampleemploying either coning and quartering method2-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. 14. Principle:It is the cheapest and most commonly used method. It makes useof heterogenous equilibrium established during the flow of asolvent(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. 15. UV CabnetTLC ScannerMicropipetteChromatogram
  16. 16. Spotting the extract:o Place 5 - 20 µl of sample as a small circular spot (< 5 mm), 1 - 2cm from the end of the TLC plate. Micropipette may be used forthe 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 tanko Keep the plate at a slight angle with the spots little above theupper level of the solvent. Due to capillary action, solvent movesupward on the plate.o Allow the solvent to travel at least about 8-10 cms
  17. 17.  Detection:o Air dry the developed plate and view in a UV cabinet undereither longwave (365 nm) or short wave (254 nm) range toidentify the fluorescing mycotoxins. In case of mycotoxinswhich do not fluoresce, spray the plate with suitable reagent (like50 % aqueous H2SO4 , Triflouro,etc). to develop fluorescence. Resolving front value (Rf):o Each mycotoxin has its characteristic color of fluorescence underUV light and a constant Rf value in a particular developingsolvent(See Table ).o Rf value is computed using the formula;
  18. 18.  Confirmation:o The presence of mycotoxin can be confirmed either by sprayingthe 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 bemeasured by using TLC Scanner / fluorodensitometer to avoidpossible human errors in comparison
  19. 19. This is an extension of TLC method. The sample spots on thedeveloped TLC plate are scraped out along with the sorbent(silica gel) and extracted with methanol for 3 minutes. Theextract is filtered and the absorbance of the filtrate is measuredin a spectrophotometer at 363 nm.This is an improvised version of TLC, where sample applicationand detection of fluorescence intensity are fully automated andcarried 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 thismethod.
  20. 20. Conical Flask100ml BeakerSpectrophotometer Linomat IVBlender
  21. 21.  Reagents:o 0.2 M NaOH (dissolve 8 g NaOH in water and make up volumeto 1 lit)o 0.41 M Ferric Chloride (dissolve 66.5 g anhydrous FeCl3 inwater 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 inwater and make up volume to 1lit)Solvents:o Acetoneo Chloroformo Developing solvent==Chloroform : Acetone : Water (88 : 12 : 1)
  22. 22. 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 waterand blend for 2 minuteso Add 150 ml acetone and blend again for 2 minuteso Filter through Whattman no.1 filter paper and transfer 75 ml offiltrate to a conical flask containing 3 g cupric carbonateo Prepare ferric gel by adding 85 ml of 0.2 M NaOH to 15 ml of0.41 M FeCl3o Add this mixture to the flask containing extract and cupriccarbonate
  23. 23. 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 thecontents slowlyCollect the chloroform layer into a 100 ml beakerAdd again 10 ml of chloroform to the separating funnel andrepeat the above step. Combine both the chloroform extractsTake 100 ml potassium wash solution in a separate separatingfunnelAdd the chloroform extract to the second separating funnel andmix it slowly
  24. 24. Collect the chloroform layer through anhydrous sodium sulfatebed 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 TLCplat along with the standardCompare the flourescence intensities of the sample and standardspots and identify the ones matching with each other2 ml of final chloroform extract is placed in the column (20 cmlength, 6 mm internal diameter with tapering end 2 mm) andeluted with chloroform : acetone (9 : 1). Aflatoxin,if present istrapped as a band above florisil layer which can be viewed underlong wave UV light as a blue fluorescent band
  25. 25. Enzyme-Linked Immuno-Sorbant Assay ELISARadioImmuno Assay IRAImmunoaffinity Column Assay ICA
  26. 26. Principle :o Antibody coated column is used to trap the mycotoxin. Thistrapped toxin is then eluted using approximate solvent andquantified in fluorometer.Equipments :o Immuno affinity columno Affinity column stand with syringeo Cuvetteo Calibrated Fluorometero Blendero Fluted filter paperReagent :oTest developero2. Methonol : water(80 : 20 by volume)o3. Mycotoxin wash buffer
  27. 27. Calibrated FluorometerImmuno affinity columnQuartz CuvetteFluted filter paperAffinity column stand with syringe
  28. 28.  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 vesselAflatoxin, Ochratoxin : 10 mlZearalenone : 1 mlDilute with purified water and mixAflatoxin, Ochratoxin : 40 mlZearalenone : 49 mlFilterRemove top cap and attach the syringe (cut 1/8 inch bottom ofcolumn )
  29. 29. Pass filtered diluted extract at the rate of 1-2 drops/secondAflatoxin : 2 mlOchratoxin, Zearalenone : 10mlPass water at the rate of 1-2 drops/secondAflatoxin, Zearalenone : 5 mlOchratoxin : First 10 ml Mycotoxin wash buffer,Later 10 ml distilled waterElute toxin in glass cuvetteAflatoxin, Zearalenone : Pass 1 ml HPLC grade methanolOchratoxin : Pass 1.5 ml Ochratoxin eluting solution.Add 1 ml of developer to the cuvette and mix wellRead in calibrated fluorometer
  30. 30. Measured by ELISAPoultry are the most tolerant of livestock species toDON. Some studies show that feeding 20-50 ppmDON has no effect on production.
  31. 31. Reagents:1. Urease enzyme solution2. Standard Urea Solutions (0, 0.5, 1, 1.5,..........5%)3. Phenol red indicator (0.1%)OrCresol 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 spotplate.Add 2-3 drops of phenol red indicator and then add 2-3 drops ofurease solution.
  32. 32. Stand for 3-5 minutesIf Urea presents solution will become red- purple in contrast tothe yellow colour of indicatorColour can be compared with the colour developed in standardsolution of varying levels of urea.white porcelein spot plate Whatman #41
  33. 33. Reagents :o 30% ammonium sulphate (dissolve 30 g (NH4)2SO4 in waterand make up volume to 100 ml)o Celite 545o Potassium wash solution (dissolve 1.12 g KOH and 10 g KCl inwater and make up volume to 1 lit)o Sodium sulphateo Silica gelo Methanol : H2SO4 ( 1 : 1 v/v )
  34. 34. Solvents :o Methanol : water (1 : 1 v/v)o Chloroformo Diethyl ethero Hexaneo Benzeneo 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
  35. 35. Developing Tank/ChamberUV cabnetPre-coated Silica Gel plateHorizontal ShakerGlass StopperedConical FlaskWhatman FP #1
  36. 36. 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 ofextract 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 intoanother separating funnelRepeat the extraction with another 10 ml of chloroform
  37. 37. More Free PowerPoint Templates at SmileTemplates.com Combine both the extracts and add 100 ml ofpotassium wash solution Swirl gently for 30 seconds and let layersseparate Drain the lower chloroform layer through abed of Sodium sulphate (in a funnel) to dryand collect 10 ml of clear filtrate Column Preparation :o Plug the bottom of a glass column ( 2x30cm )with glass wool and add 5 g anhydroussodium sulphate. Fill the column to half levelwith chloroform and add 10 g silica gel.
  38. 38. More Free PowerPoint Templates at SmileTemplates.como Wash sides of column with chloroform andstir to eliminate air bubbles.o Drain off chloroform leaving about 7 cmabove the upper level of silica gel. Add 15 ganhydrous sodium sulphate withoutdisturbing the silica gel.o Drain off chloroform to the upper level ofsodium sulphate Wash the column serially with 50 ml ofdiethyl ether and 10 ml of chloroform anddiscard the washings
  39. 39. More Free PowerPoint Templates at SmileTemplates.com Mix 10 ml of sample extract with 30 ml ofhexane and add to the column and slowlydrain until solvent is about 1 cm aboveSodium sulphate Add in succession 30 ml benzene and 40 mlacetone : benzene (5: 95) and discard boththe washings Elute T-2 with diethyl ether until 30 ml ofeluate is collected and evaporate the eluate
  40. 40. More Free PowerPoint Templates at SmileTemplates.com Dissolve the residue in 0.5-1.0 ml diethylether. Spot on TLC along with the standard(5-20 µL or any other suitable range) anddevelop the plate in toluene : ethyl acetate :formic acid (6 : 3 : 1) Air dry the plate and spray withmethanol:H2SO4 (1:1) Dry at 110 C for 10 minutes Observe blue fluorescence under long waveUV light (365 nm) Compare the intensities of the blue fluorescent spotsof the sample with those of standard and identify theones matching each other
  41. 41. References:o Commercial Poultry Nutritiono Analysis of Toxins by layer chromatography by R.W. Nicol & R.C. Sinhao Laboratory Manual on Laboratory Control of Animal feed by Dr.G.Devegowdao Lecture of Dr. Naeem Tahir (PMAS Arid AgricultureUniversity, Rawalpindi)o Simple Sorgham Grain test for tannin by John R.N Tayloro www.foa.como www.hamletprotein.como www.allaboutfeed.neto www.poultryhub.org