Acid Detergent Fiber Determinations (ADF)

Reagents

  1. ADS - ACID DETERGENT SOLUTION - Weigh 400 g Hexadecyltrimethyl-ammonium bromide in a 2 L beaker under the hood. (This material is harmful if inhaled or absorbed through the skin so a respirator and gloves should be worn while making this solution.) Add deionized water to this beaker and stir until mixture can be poured. Transfer this solution to a 4 L beaker.  Bring the volume up to about 3 L with water  and stir thoroughly with a long stirring rod. Very slowly add 556 mL conc. H2S04 to the solution and continue to stir vigorously with the stirring rod. (If the acid is poured in too quickly without stirring, the solution may turn brown. If this happens add a little more water and continue to stir.) At this point there may still be some undissolved material, so place the beaker on a heater/stirrer and stir with heat until the material is dissolved. Transfer this solution to a 20 L carboy and dilute to 20 L with deionized H20. This may be purchased as a concentrate or straight solution.
  2. ACETONE

Apparatus

  1. ANKOM FIBER ANALYZER #F200
  2. 3 3000 mL BEAKERS 
  3. HOT PLATE
  4. FILTER BAGS (ANKOM CO. #F57)  
  5. HEAT SEALER #1915
  6. BAG HOLDER

Procedure

  1. Prepare Filter Bags/Samples.
    1. Weigh labeled filter bag (ANKOM Co. #F57). Record weight.  
    2. Tare bag holder containing sample bag.  Weigh 0.5 g (+0.01 g) of sample  into bag. Record weight of sample.
    3. Seal the bag closed within 1 cm from the open edge using the heat sealer.
    4. Spread sample uniformly inside the filter bag. This should be done by shaking and tapping the bag to eliminate clumping. A uniform distribution of the sample inside the bag is very important to achieving good precision and accuracy in the analysis.
    5. Place 24 bags in the bag suspender. The bag suspender is composed of 9 individual baskets and a center post. The 9th basket acts as a top of the 8th. Place three bags in each basket (separated 120 degrees apart). Stack baskets on center post ensuring bags on different levels are not positioned directly above each other in order to maximize the flow of solution around each bag. (Run a check with each batch.)
  2. Fill pressure vessel with 2000 mL of ambient temperature AD or ND solution. Less bags and solution can be used per study but a minimum of 1500 mL of solution has to be used in the digestion vessel. (See note below*.)
  3. Place bag suspender with samples into the solution in vessel. Place weight on top of samples.
  4. Start timer (60 min. for ADF and 75 min. for NDF), screw down vessel top and turn agitation and heat on.
  5. While samples are being digested heat 9 L of rinse water to 90-100oC.
  6. Turn heat and agitation off when indicated by timer, slowly open the valve and exhaust hot solution before opening lid. NOTE: THE SOLUTION IN THE DIGESTION VESSEL IS UNDER PRESSURE. THEREFORE, THE VALVE NEEDS TO BE OPENED FIRST IN ORDER TO RELEASE THE PRESSURE BEFORE THE VESSEL LID CAN BE OPENED.
  7. After the solution has been exhausted close valve and open the lid. Add approximately 2 L of hot rinse water. Lower lid and turn agitate on. Repeat hot water  rinse three more times. For NDF assays add 4 mL of heat stable alpha-amylase  (ANKOM Co. #FAA) to each of the first two rinses (8 mL total). Add amylase to  beaker to mix before adding solution to vat.
  8. 8. Remove filter bags from bag suspender and gently press excess water out. Place bags in a small beaker and pour in enough acetone (240 mL) to cover bags. Allow bags to soak 3 min. Remove and lightly press excess acetone out.
  9. 9. Spread bags out and let air dry. Complete drying in oven (105oC) overnight.
  10. Transfer samples to plastic bags. Take one fiber bag out at a time and close plastic bag with remaining samples to exclude moisture. Weigh. Record weight.
  11. Calculate percent fiber: % Fiber = 100(C-(A*D))/B
    Where    :
    • A = Bag tare weight
    • B = Sample weight
    • C = Final bag weight after digestion and drying
    • D = Blank bag correction (final oven dried weight/original blank bag weight)
    •      (use value of D=0.997)
    *NOTE ADF
    • < 6 samples - hold until next day
    • 7-15 samples - use 1500 mL ADF
    • 16 samples - use 1600 mL ADF
    • 17 samples - use 1700 mL ADF
    • 18 samples - use 1800 mL ADF
    • 19 samples - use 1900 mL ADF
    • 20-24 samples - use 2000 mL ADF
  12. Record values with one decimal.

Quality Control

  1. Run an NFTA check sample with each batch.

References

  1. Goering, H. K., and P. J. Van Soest. 1970. Forage Fiber Analysis (Apparatus, Reagents, Procedures, and Some Applications). Agri. Handbook, No. 379, ARS, USDA.