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Dairy One Forage Lab Analytical Procedures -
February 2007
I. Dry Matter
A.
B.
C.
D. 		Oven – 60ºC for 4 hours (forced air).
Oven – 135ºC for 2 hours - AOAC 930.15.
Oven – 105ºC for 3 hours - NFTA Method 2.1.4.
Near Infrared Reflectance Spectroscopy (NIRS) – AOAC 991.01.
II. Protein
A. Crude Protein (CP)
1. Kjeldahl – AOAC 984.13. 10.5g of catalyst (ratio of 10g K2SO4 to 0.3g CuS04) is used. A Boric acid receiving solution contains methyl red-methylene blue indicator.
2. Block Digestion and Tecator Kjeltec 2300 or 2400 Analyzer – Modified Kjeldahl procedure with automatic distillation and titration.
a.
 AOAC 2001.11.
b. FOSS Tecator, Application Note AN 300, pp. 1-12, 1987 "The Determination of Nitrogen according to Kjeldahl using Block Digestion and Steam Distillation".
3. Leco FP-528 Combustion analyzer.
a. AOAC 990.03.
b. Leco Application Note "Nitrogen/Protein in Animal Feeds" Form 203-821-146, 2000.
4. NIRS – Foss NIR Systems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
 

Used for grass, grass-legume mixtures, legume hays, haylages, fresh forages and pastures; corn silage and corn stalks (fresh and fermented); corn silage and haylage mixtures; shelled, ear, and snaplage corn; hays, fresh forages, pastures and silages for barley, wheat, oats, triticale, peavine, soybean and triticale and peas; fresh forages and silages for rye, sorghum, sorghum-sudan and sudangrass; barley, oats, triticale and wheat grains; brewers grains; distillers grains; total mixed rations (TMR's).
B. Soluble Protein (SP)
1. Cornell Sodium Borate-Sodium Phosphate Buffer Procedure. Soy products incubated at 39°C. All other samples incubated at ambient temperature. Cornell Nutrition Conference Proceedings, 1990, pp. 85-86.
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
C. Degradable Protein (Rumen Degradable Protein (RDP))
1. Cornell Streptomyces griseus (SGP) enzymatic digestion.
Enzyme concentration held constant.

a.

Concentrates incubated for 18 hrs. Cornell Nutrition Conference Proceedings, 1990. pp. 81-88.
b. Forage samples incubated for 2 hrs at higher SGP concentration. J. Dairy Sci. 1999. 82: 343-354.
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
D.

Acid Detergent or Neutral Detergent Insoluble Crude Protein (ADI-CP, NDI-CP)
1. ADF or NDF residue is subjected to Kjeldahl or Kjeltec analysis to determine the protein fraction bound to the fiber. Sodium Sulfite not used for NDI-CP.
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 - AOAC 989.03.
E. Non Protein Nitrogen (NPN; reported as crude protein equivalent – CPE)
    1. Urea and Ammoniacal Nitrogen – AOAC 941.04.
2. Urea – AOAC 967.07.
3. NIRS – Ammonia – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03. Analyzed on corn silage and fermented
haycrop forages only.
III. Fiber
A. Acid Detergent Fiber (ADF)
1. ANKOM A200 Filter Bag Technique (FBT). ANKOM Application Note 01/02 "Method for Determining Acid Detergent Fiber". Solutions same as AOAC 973.18 (C). Samples individually weighed into filter bags and digested for 75 minutes as a group of 24 in 2L of ADF solution in ANKOM A200 Digestion Unit. FBT eliminates filter steps. Samples are rinsed three times with boiling water in filter bags followed by an acetone rinse and drying at 100ºC for 2 hours.
2. Liquid samples – AOAC 973.18 (C). Whatman 541 filter paper and buchner funnels.
3. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
B. Neutral Detergent Fiber (NDF)
1. ANKOM A200 Filter Bag Technique (FBT). ANKOM Application Note 01/02 "Method for Determining Neutral Detergent Fiber (aNDF)". Solutions same as Journal of Dairy Science 74:3583 - 3597. Samples individually weighed into filter bags and digested for 75 minutes as a group of 24 in 2L of NDF solution in ANKOM A200 Digestion Unit. Four ml of Alpha Amylase and 20g sodium sulfite are added at the start of digestion. FBT eliminates filter steps. Samples are rinsed three times with boiling water. Alpha Amylase is added to the first 2 rinses. Water rinses are followed by an acetone rinse and drying at 100ºC for 2 hours.
2. Liquid samples – Journal of Dairy Science 74:3583 - 3597, 10/91.
3. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
C. Crude Fiber – AOAC 962.09
Samples filtered through bingham linen after first boil and through Whatman AH-934 glass membrane filter paper after second boil.
D. Lignin
1. ANKOM A200 Filter Bag Technique (FBT). ANKOM Application Note 01/02 "Method for Determining Acid Detergent Lignin in DaisyII Incubator". Solution same as AOAC 973.18(D). ADF performed as in III.A.1. ADF residue digested as a group of 24 in 72% w/w sulfuric acid for 3 hours in ANKOM DaisyII Incubator at ambient temperature.
2. Liquid samples – AOAC 973.18(D). No asbestos.
3. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
E. In-Vitro True Digestibility (IVTD / NDFD)
1. ANKOM DaisyII Filter Bag Technique (FBT). ANKOM Application Note 11/00 "In Vitro True Digestibility using the DaisyII Incubator". Rumen fluid collected from TMR fed, high producing lactating cow. Feed samples incubated in Van Soest buffer/rumen fluid mixture for 24, 30, or 48 hours under anaerobic conditions at 39ºC. After incubation, samples extracted using NDF procedure to remove bacterial contamination. Residue is undigested fibrous material and is used to determine in-vitro true digestibility (IVTD) and neutral detergent fiber digestibility (NDFD).
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
IV. Minerals
A. Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, Co

Analyzed using a Thermo Jarrell Ash IRIS Advantage HX Inductively Coupled Plasma (ICP) Radial Spectrometer.
1. General Feeds and Forage Types – Thermo Jarrell Ash "The Spectroscopist" Dec. 1994, Vol. 3. No. 1. pp 6-9. Samples ashed in muffle furnace at 500ºC for 4 hours. Three ml of 6N HCl are added to ash residue and evaporated to dryness on a 100º - 120ºC hot plate. Minerals extracted with acid solution (1.5N HNO3 + 0.5N HCl) and determined using an IRIS Advantage HX.
2. Grain and Mineral Mixes – High Organic Matter (OM) mixes ashed 2 hours at 500ºC. Low OM samples not ashed. 10 ml Mineral Mix extracting solution (1.8N HCl + 0.3N HNO3) added to sample and digested on 100º - 120ºC hot plate. Filtered through Whatman 4 filter paper into volumetric flasks using 1.5N HNO3 + 0.5N HCl and minerals determined using an IRIS Advantage.
3. NIRS – (Ca, P, Mg, K) – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
B. Sulfur (S)
1. Leco Model SC-432. Leco Application Note "Sulfur in Plant Tissue" Form 203-601-229, 08/92. Samples combusted in oxygen rich atmosphere at 1350°C. Sulfur bearing compounds break down freeing sulfur, then oxidized to form SO2. Gases flow through an infrared detection cell which measures the concentration of SO2. The instrument converts that value and reports a percent sulfur.
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
C. Chloride Ion (Cl)
1. Brinkman Metrohm 716 Titrino Titration Unit – 0.5g dried, ground sample extracted in 50ml 0.1N HNO3 followed by potentiometric titration with AgNO3 using Brinkman Metrohm 716 Titrino Titration Unit with silver electrode.
a. Metrohm Application Bulletin No. 130 by Metrohm Ltd., C-H-9101 Herisau, Switzerland distributed in the US by Brinkmann Instruments Inc., One Cantiaque Road, PO Box 1019, Westbury, NY 11590-0207, phone 1-800-645-30502.
b. The method by Metrohm is similar to the concepts found in: Cantliffe, D.J., MacDonald, G.E. and Peck, N.H. 1970. The potentiometric determination of nitrate and chloride in plant tissue. New York's Food and Life Sciences Bulletin. No.3, September 1970. Plant Sciences. Vegetable Crops Geneva. No. 1: 5-7.
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
V. Supplemental Services
A. Crude Fat
1. Ether Extraction – AOAC 2003.05. Crude Fat in Feeds, Cereal Grains, and Forages.
Extraction by Soxtec HT6 System using anhydrous diethyl ether. Crude fat residue determined gravimetrically after drying.
2. Acid Hydrolysis – AOAC 954.02 - Crude Fat in Pet Food.
3. Dried Milk – AOAC 932.06 A (b) and 932.06 B (Roese-Gottlieb Method).
4. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
B. Ash
1. AOAC Method 942.05.
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
C.

Nitrates (%NO3 or ppm NO3-N)

RQflex® Reflectometer Method.

EMD Application Note "Nitrates in Plant Sap", NITRA18.emd; 07/99; extraction modified. EMD Chemicals Inc., 480 S. Democrat Road, Gibbstown, NJ 08027.

1g of dried, ground sample is extracted in 50ml deionized water for 20 minutes by shaking at 280 oscillations/minute. Samples are filtered through Whatman 934-AH (1.5um) filter paper, then analyzed by RQflex® Reflectometer using Relectoquant® Nitrate test strips.

When a test strip is immersed in a sample, a reducing agent reduces nitrate ions to nitrite ions. In the presence of an acidic buffer, the nitrite ions react with an aromatic amine to form a diazonium salt. The salt reacts with N-(1-naphthyl)-ethyelene-diamine to form a red-violet azo dye that is measured reflectometrically. Nitrate concentration is proportional to the color reaction.

Each strip contains two reaction zones generating dual replicate analyses per sample. The RQflex® Reflectometer's double optic system measures the analyte concentration based on the light reflected from the dual reaction zones. Barcode-controlled software
calculates the mean of those two measurements.
D. pH

15g wet sample placed into 250-ml beaker. 200ml deionized water added, stirred, and allowed to stabilize five minutes. Analyzed using Thermo Orion Posi-pHlo SympHony Electrode and Thermo Orion 410A meter.

Calibrated with buffers referenced to NIST SRMs. pH 4 butter contains potassium hydrogen phthalate and pH 7 buffer contains sodium phosphate dibasic and potassium phosphate monobasic.
E. Starch
1. YSI 2700 SELECT Biochemistry Analyzer – YSI Incorporated, Application Note Number 319.

Samples are pre-extracted for sugar by incubation in water bath and filtration on Whatman 41 filter paper. Residues are thermally solubilized using an autoclave, then incubated with glucoamylase enzyme to hydrolyze starch to produce dextrose. Samples injected into sample chamber of YSI Analyzer where dextrose diffuses into a membrane containing glucose oxidase. The dextrose is immediately oxidized to hydrogen peroxide and D-glucono-4-lactone. The hydrogen peroxide is detected amperometrically at the platinum electrode surface. The current flow at the electrode is directly proportional to the hydrogen peroxide concentration, and hence to the dextrose concentration. Starch is determined by multiplying dextrose by 0.9.
2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
F. Water Soluble Carbohydrates (WSC)
1. West Virginia University Procedure by W.H. Hoover and T.K. Miller Webster. Determination of Nonstructural Carbohydrates.

Samples incubated with water in a 40°C bath extracting water soluble carbohydrates comprised of simple sugars and fructan. WSC determined after acid hydrolysis with sulfuric acid and colorimetric reaction with potassium ferricyanide.

Hall, M.B., W.H. Hoover, J.P. Jennings and T.K. Miller Webster. 1999. A method for partitioning neutral detergent soluble carbohydrates. J. Sci. Food Agric. 79: p.2081.

2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
G. Ethanol Soluble Carbohydrates (ESC)
1.







2.		 Hall, M.B., W.H. Hoover, J.P. Jennings and T.K. Miller Webster. 1999. A method for partitioning neutral detergent soluble carbohydrates. J. Sci. Food Agric. 79: p.2079-2086.

Samples shaken with 80% ethanol to extract ethanol soluble carbohydrates comprised of simple sugars. ESC determined colorimetrically using a phenol-sulfuric acid reaction.

NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.
H. Volatile Fatty Acids (VFA)
1. Gas Chromatography and Biochemistry Analyzer methods.

Extraction – Samples blended for 2 min. in deionized water, filtered through cheesecloth, then filtered through disposable syringe filter. Adapted from Personal Communication, L.E. Chase, Ph.D., Cornell University.

Analysis –
a. Acetic, Propionic, Butyric, Iso-butyric acids – Aliquot of extract mixed 1:1 ratio with 0.06M Oxalic acid containing 100ppm Trimethylacetic acid (internal standard). Samples injected into a Perkin Elmer Autosystem XL Gas Chrmatograph containing a Supelco packed column with the following specifications: 2m x 2mm Tightspec ID, 4% Carbowax 20M on 80/120 B-DA.

Procedure based upon:
"GC Separation of VFA C2-C5" Supelco GC Bulletin 749F, 1975.
"Analyzing Fatty Acids by Packed Column Gas Chromatography" Supelco GC Bulletin 856A, 1990.
"Volatile Fatty Acid SOP" W.H. Miner Institute, Chazy, NY.
b. Lactic acid - Aliquot of extract analyzed for L-Lactate using a YSI 2700 SELECT Biochemistry Analyzer equipped with an L-Lactate membrane. YSI User's Manual, page 4-7.

Samples injected into sample chamber of YSI Analyzer where L-Lactate diffuses into a membrane containing L-Lactate oxidase. The L-Lactate is immediately oxidized to hydrogen peroxide and pyruvate. The hydrogen peroxide is detected amperometrically at the platinum electrode surface. The current flow at the electrode is directly proportional to the hydrogen peroxide concentration, and hence to the L-Lactate concentration. Total Lactic acid is determined by multiplying L-Lactate by 2.0.

2. NIRS – Foss NIRSystems Model 6500 with Win ISI II v1.5 – AOAC 989.03.

Lactic and Acetic acids only. Analyzed on corn silage and fermented haycrop forages only.
VI. Manure
All results corrected for density except total solids.
A. Total Solids
1. Liquid or solid; no bedding.
  • Oven - 100°C for 16 hours (gravity).
2. Liquid or solid with bedding.
  • Oven - 60°C for 6-8 hours (forced air).
  • Near Infrared Reflectance Spectroscopy - AOAC 991.01.
B. Nitrogen, total
1. Kjeldahl - AOAC 984.13 - for wet samples or as-is basis (primary).
2. Kjeltec - AOAC 2001.11 - for wet samples or as-is basis (secondary).
3. Combustion by Leco FP-528 - AOAC 990.03 - for pre-dried samples.
C. Ammonia-Nitrogen
1. Distillation - AOAC 941.04.
D. Organic Nitrogen
1. by difference (Total N - Ammonia-N).
E. Minerals
1. Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo, Co
  • Weigh 1g dried, ground or 3g wet sample. Dry ash at 500°C for 4 hours, followed by wet ash with 1:1 HCl on 100 - 120°C hot plate. Final extraction and dilution in 1.5N HCl + 0.5N HNO3.
  • Analysis by Thermo IRIS Advantage HX Inductively Coupled Plasma (ICP) Radial Spectrometer.
2. Sulfur (S)
Combustion by Leco Model SC-432. 200mg dried sample burned in oxygen rich atmosphere at 1350°C. Sulfur bearing compounds are broken down freeing sulfur, then oxidized to form SO2. Infrared detection cell measures SO2 concentration and instrument converts and reports %sulfur.
3. Chloride (Cl)
  • Weigh 0.5g dried, ground or 5g wet sample. Extract in 50ml 0.1N HNO3 followed by potentiometric titration with AgNO3 using Brinkmann Metrohm 716 Titrino Titration Unit with silver electrode.
F. Ash
1. AOAC 942.05.
G. pH
1. AOAC 973.04 (Technique similar to pH of peat).
35ml liquid sample poured into 50ml beaker. 15g solid or semi-solid sample weighed into 200 ml deionized water, stirred, and allowed to stabilize five minutes. Analyzed using Thermo Orion Posi-pHlo SympHony Electrode and Thermo Orion 410 A meter.
H. Nitrates
1. RQflex Reflectometer Method.
Weigh 1g of dried, ground or 10g wet sample. Extract in 50ml deionized water for 20 minutes by shaking at 280 oscillations/minute. Filter through Whatman 934-AH (1.5um) filter paper, then analyze by RQflex® Reflectometer using Relectoquant® Nitrate test strips.
I. Density
1. Standard Vial Method.
Samples weighed into fixed volume vessel. Density calculated and expressed in kg/l, lbs./ft3, and lbs./gal.
VII. Water
A. Coliform and E. coli (presence/absence in 100ml)
Colilert® -IDEXX Laboratories, Inc., One IDEXX Drive, Westbrook, Maine 04092, Colilert® uses the patented Defined Substrate Technology® (DST®) to simultaneously detect total coliforms and E. coli. Two nutrient-indicators, ONPG and MUG, are the major sources of carbon in Colilert® and can be metabolized by the coliform enzyme ß-galactosidase and the E. coli enzyme ß-glucuronidase, respectively.

As coliforms grow in Colilert®, they use ß-galactosidase to metabolize ONPG and change it from colorless to yellow to indicate presence.

E. coli use ß-glucuronidase to metabolize MUG and create fluorescence to indicate presence. Since most non-coliforms do not have these enzymes, they are unable to grow and interfere.

Colilert® is US FDA Approved for Dairy Waters.
Milk Laboratory Evaluation Form FDA 2400m (3/01).

Colilert® is also US EPA-approved for drinking water presence/absence(P/A) and Most Probable Number (MPN) and for source water. Pertinent references:
June 29, 1989 US EPA Federal Register Colilert® coliform approval.
June 10, 1992 US EPA Federal Register Colilert® E. coli approval.

Colilert® detects total coliforms and E. coli at 1 organism/100 ml.
B. Standard Plate Count (colonies per ml)

US FDA Milk Laboratory Evaluation Form FDA 2400a (1/01).

Petrifilm Aerobic Count Method – Deposit 1ml of sample onto petrifilm and cover. Distribute sample with spreader and allow gel to solidify for 1 minute. Incubate 48 hours at 32°C. Count colonies when incubation time is complete.

C. pH

AOAC 973.41

Analyzed using Thermo Orion Posi-pHlo SympHony Electrode and Thermo Orion 410 A meter. Calibrated with buffers referenced to NIST SRMs. pH 4 buffer contains potassium hydrogen phthalate and pH 7 buffer contains sodium phosphate dibasic and potassium phosphate monobasic.

D. Nitrates (ppm NO3 and ppm NO3-N)

RQflex Reflectometer Method. Nitrate in Waste Water. NITRA12.emd; 12/95.

EMD Chemicals Inc., 480 S. Democrat Road, Gibbstown, NJ 08027.

Reflectometric determination after reduction to nitrite and reaction with Griess reagent. Reflectoquant® Nitrate test strip immersed in water, allowed to react for specified time, then analyzed by RQflex® Reflectometer.

Nitrate-Nitrogen (NO3-N) calculated as Nitrates (NO3) divided by 4.427.

E. Sulfates (ppm SO4 and SO4-S)
Turbidimetric Method. 957-13-3. (Based upon principles in AOAC 973.57).

Orbeco Analytical Systems, Inc., 185 Marine Street, Farmingdale, NY 11735, 1-800-922-5242.

Conditioning reagent containing glycerol, alcohol, sodium chloride and hydrochloric acid added to water. BaCl2 crystal added resulting in precipitation of sulfate as BaSO4. Suspension is measured photometrically at 420nm and sulfate concentration determined from a standard conversion table.

Sulfate-sulfur (SO4-S) calculated as sulfates (SO4) divided by 2.996.
F. Total Dissolved Solids (ppm TDS)
Conductivity Method. ES&D Model 76 Conductivity meter.

The total quantity of free ions is determined by ability of the sample to conduct an electrical current. Immerse electrode in water while gently stirring. Measure temperature of water, set temperature knob, allow meter to stabilize 15 seconds, then record reading.

G. Minerals
1. ppm Ca, P, Mg, K, Na, Fe, Zn, Cu, Mn, Mo
Analyzed directly with no sample preparation by Thermo IRIS Advantage HX Inductively Coupled Plasma (ICP) Radial Spectrometer.

ppm Hardness as CaCO3 equivalent calculated as (Ca x 2.5) + (Mg x 4.1).
2. ppm Chloride (Cl)
25ml 0.2N HNO3 added to 25ml of water followed by potentiometric titration with AgNO3 using Brinkmann Metrohm 716 Titrino Titration Unit with silver electrode.


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