<< Appendix E - Taking a Good Forage Sample for Analysis

 


Appendix F - Critical Conditions in Determining Detergent Fibers

References:
Mertens, D.R. 1992. Critical conditions in determining detergent fibers. Proc. NFTA Forage Analysis Workshop, Sept. 16-17, Denver, CO. pp. C1-C8.

Abstract

Fiber is a uniquely nutritional entity that we attempt to measure using chemical solubility methods. Because there is no direct relationship between solubility and nutritional availability, the method used to isolate fiber, in effect, defines it. This indicates that fiber methods must be followed exactly to obtain values that are valid and reproducible. Unfortunately laboratories sometimes modify fiber methods for convenience or speed without understanding the effects these changes have upon results. Some of the conditions and steps in the detergent fiber methods are critical in obtaining accurate results. Among these are: subsampling, drying, grinding (sample particle size), sample amount, standardization of reagents, removal of starch and nitrogen contamination, timing and temperature of refluxing, transferring residues, washing fibrous residues, type of filtration vessel and weighing method. Some types of samples are difficult to filter in the NDF procedure, and modifications are proposed that aid the filtering of starch-, pectin- and fat-containing samples.


Introduction

Fiber is a uniquely nutritional term that can be defined as the indigestible or slowly-digesting components of feeds that occupy space in the gastrointestinal tract of animals. Chemically these components are a variable mixture of cellulose, hemicelluloses, some pectins and lignin, with indigestible proteins and lipids. This nutritional definition of fiber indicates that it can truly be measured only by the animal. However, nutritionists need a practical and routine way of measuring fiber and must compromise between the theoretical concept of fiber and the utility of using chemical solubility to isolate and measure fractions that closely resemble the nutritionally defined fraction called fiber. Because there is no guarantee of direct correspondence between chemical solubility and nutritional availability, in reality, fiber is defined by the method used to isolate it. The actual definition of fiber becomes method dependent, which explains why there are so many different fiber analyses (crude fiber, acid detergent fiber, neutral detergent fiber, amylase-neutral detergent fiber, dietary fiber, etc.).

The abundance of fiber methods is further complicated by the modifications of each method that are commonly used. Sometimes these modifications are developed to meet the specific needs of a particular application or research project. Other times modifications are made for convenience or to increase the speed of fiber analysis. Because fiber is defined by the method used to isolate it, it should be clear that modifications have the potential for defining a new fiber value that is not comparable with the parent method. The sensitivity of fiber values to method suggests that fiber methods must be followed exactly to be reproducible. To be acceptable, any modification of fiber methods must be evaluated thoroughly with several feed samples representing each type of feed ingredient. The objective of this paper is to discuss some of the critical steps and conditions in detergent fiber analyses and indicate the potential problems inherent in modifying these methods.

Factors Causing Variation in Detergent Fiber Analyses

Subsampling and Segregation. In general, the fiber content of large particles is greater than that of small particles for most feed ingredients. Thus, any process that segregates a sample by size (such as shaking during shipment, grinding or grab sampling) can lead to a subsample that differs from the true average of the sample. One of the most insidious segregation processes is grinding. The tough, large particles that are retained in the grinder and finally pulverized to the extent they pass through the screen are high in fiber. If they are brushed or vacuumed from the mill and not included in the sample, there is an error. If they are ground until they pass, but the sample is not mixed thoroughly after grinding, there is an error because the last material (high fiber) out of the mill is on the top of the sample due to grinder segregation of the sample.

Sample Drying. Proteins and carbohydrates can form insoluble compounds (Maillard or browning products) when exposed to high temperatures in the presence of moisture. These Maillard products are measured as artifact fiber and lignin in the detergent system. Thus, feed samples should never be exposed to temperatures above 60C (140F) during drying and a maximum of 50C (125F) is preferred.

Sample Grinding. Fiber methods function by extracting and solubilizing non-fibrous compounds from feed particles. It is expected that extraction efficiency should increase as the size of particles decreases because reagents and washing solvents have less matrix to penetrate. Furthermore, fibrous residues are filtered on coarse porosity membranes suggesting that fine fiber particles may be washed out of the residue or plug the filter membrane. These factors explain why finer grinding of feed samples results in lower fiber values. However, a compromise is necessary between fine grinding to increase extraction efficiency and coarse grinding to prevent lost of fiber particles and plugging of the filtration vessel. The recommended grinding procedure uses a Wiley mill with a 1 mm screen. Cyclone mills generate particle size distributions that are smaller than Wiley mills when similar size screens are used because cyclone mills force the particles through the screen at an angle. Using the same size screen, Udy cyclone mills will produce an average particle size that is one half that of Wiley mills resulting in slightly lower fiber values and greater filtering difficulties during detergent analyses.

Standardizing reagents. To provide accurate ADF and NDF values, both reagents should be standardized. Measurement of ADF depends on the use of 1 N sulfuric acid. Normality of the acid used for acid detergent (AD) solution should be verified by titrating it against a primary base standard. We use TRIS or THAM (Tris- hydroxymethyl-aminomethane) as a primary base standard. Make a .9-1.0 N solution of THAM (weigh and calculate normality to 4 decimal places) and use it in the buret. Titrate (to the nearest .01 ml) 10 ml of acid in a beaker with stirring using a pH indicator that changes near pH 7.0. If AD solution is not between .99 and 1.01 N, adjust normality by adding water or concentrated sulfuric acid.

Neutral detergent (ND) solution should be standardized to a pH of 6.9 to 7.1 (I prefer a narrower range of 6.95 to 7.05). If pH differs by more than .2 from 7.0, check reagents to make sure the wrong chemicals were not used and consider discarding the neutral detergent solution. If pH is between 6.8 and 7.2, adjust pH by adding either HCl or NaOH to obtain a pH of 7.00. The original ND solution developed by Van Soest contained 2-ethoxyethanol which has been found to be toxic. Triethylene glycol should be substituted for it on an equal volume basis. This chemical should not be eliminated from ND solution because it aids in the removal of non-fibrous residues from some feeds. Amylase solution should be standardized so that 2 ml of enzyme solution added at boiling and during the first filtration step removes all traces of corn starch from the fritted disk of Gooch crucibles. Make sure that 0.5 gm of sodium sulfite is added to each sample before refluxing. It is important for the removal of protein from NDF and is especially critical in the removal of nitrogenous contamination from cooked or heated feeds, animal byproduct feeds and fecal or digesta samples.

Sample Amount. The ratio of sample to detergent solution can have a small, but significant, effect on fiber analyses. The standard procedure for ADF uses 1.0 g of sample with 100 ml of AD solution, while the NDF procedure uses 0.5 g of sample in 50 ml of ND solution. The selection of sample amount to analyze is a compromise among extraction efficiency, reagent cost and weighing errors. Larger sample amounts increase reagent costs when maintaining the same sample:solution ratio. Smaller sample amounts magnify any weighing errors, e.g., if the residue weighs .0100 g with a weighing error of .0002 g, the error is 2%; however, if the residue weighs only .0020 g, the same error is 10%. Since ADF has smaller residues and the residue is often used for lignin analysis, a larger sample amount is recommended for ADF.

Pre-soaking of Samples Before Refluxing. Some laboratories weigh and add AD or ND solutions to the samples the night before analysis. This is NOT recommended. AD is a strong acid and it begins to degrade some fiber components when exposed to the sample for prolonged times. Sodium sulfite makes the ND solution unstable (that is why it is not added to the solution) and soaking in ND increases extraction efficiency. ALWAYS add detergent reagents at the time of refluxing.

Varying Refluxing Times and Temperatures. Extraction of detergent fibers is both time and temperature dependent. As the time and temperature increase, the amount of fibrous residue recovered decreases. It is critical to each method, especially ADF, that the time of refluxing be 60 minutes from the time of boiling. Refluxing should be at a temperature that causes a rolling agitation of feed particles. We calibrate our heating elements so they will bring 100 ml of water at room temperature to a boil in 3 to 4 minutes. This setting is marked on the heater and used during fiber refluxing. Incomplete Transfer of Residues to the Crucible. The greatest source of poor technique is the loss or incomplete transfer of all fibrous residue from the Berzelius beaker to the Gooch crucible. Sometimes residues are stuck to the sides or bottom of the beaker. These residues must be policed free before they can be transferred. At other times the last drop from the beaker is allowed to flow down the outside of the vessel when it is turned upright after pouring contents into the crucible. We recommend that the beaker be kept inverted over the crucible and be rinsed with a fine stream of hot water to transfer all particles. If the beaker must be turned upright during transfer, be sure to wipe the last drop from the lip of the beaker onto the lip of the crucible. Often this last drop contains significant fiber because particles have settled in the beaker during transfer. Transfer should be so complete that beakers do not need to be washed between uses. Beakers should be checked routinely for cleanliness to insure that previous transfers were complete.

Washing Residues with Hot Water and Acetone. The most common error made by fiber analysts is incomplete washing of fiber residues to remove detergent and soluble feed components. All too often residues are rinsed, rather than soaked during the washing steps. Feed particles are filled with voids that can trap solutions and components. These voids cannot be washed free of contaminants by simply rinsing the outside of the particle. The laws of mass action must be used to equilibrate the liquids within the void with clean wash water on the outside of the particle. This is a time dependent process. Thus, fibrous residues must be soaked in 30-40 ml of clean hot water (95-100C) at least 2 minutes each time to remove the detergent and soluble compounds trapped in the voids of particles. The larger the volume of water and the longer the time of soaking, the more complete will be the extraction of soluble contaminants of fiber. We normally extract with 40 ml of water for 5 minutes.

The same principles are true for acetone washes which are used to remove residual lipids (fats) from the sample. Simply washing the outside of particles with acetone will not extract all the lipid. Both the time and amount of clean acetone are important. A minimum of 20 ml of acetone for 2 minutes is needed and we normally use 30 ml extracted for 5 minutes.

It is especially important that all traces of acid be washed from ADF residues and filtration vessels. With crucibles it is desirable to rinse the underside of the crucible, and with filter paper it is wise to rinse the edges of the paper. If residual acid remains, it will migrate to the edges of particles and become concentrated during drying. The concentrated acid will char the fiber or filter paper at 100C. Charring signifies oxidation and loss of organic matter resulting in low weights. In crucibles only the fiber is lost and low fiber values are obtained. When filter paper is used, the loss of weight can be from both fiber and paper. Because the acid migrates to the edge of the paper, mostly paper weight is lost resulting in high fiber values.

Filtration. Several factors are important in making filtration of fiber residues effective and efficient. Normally minimum filtration vacuum should be used to prevent plugging the filter membrane with fiber residues and losing fine particle. The vacuum source should be constant and have reserve capacity. Some laboratories successfully use water aspirators, but we have not found them acceptable in our lab. We use a two-stage diaphragm pump with two 18 liter glass reservoirs as waste traps and vacuum reserves. It is also important that the manifold and vacuum lines be constructed to minimize the trapping of foam. Foam will greatly reduce the effective vacuum at the crucible.

We have designed a manifold that minimizes vacuum leaks and foam in the system, yet is durable and economical to construct. The manifold is designed for Gooch crucibles, but can easily be modified for use with Buchner funnels or paper funnels. The basic design fits crucibles tightly and allows back flushing of problem crucibles by removing and reinserting them into the holder.

The choice of filtration vessel is a compromise between filtration ease and fiber recovery. Coarse membranes will allow some fine fiber particles to be lost, but fine membranes often plug, making filtration difficult. The retention size of some common filtration vessels indicates the potential variation that can occur:

Vessel or Membrane Retention Size
(micrometers)
Extra coarse fritted disk, Gooch crucibles 170-220
FiberTec P0 special crucible 160-250
FiberTec P1 special crucible 90-150
California Buchner funnel with 200 mesh screen 70-85
FiberTec P2 standard crucible 40-90
Coarse fritted disk, Gooch crucible (50 ml)* 40-60
FiberTec P3 special crucible 14-40
Whatman 41/54/541 filter paper 20-25
Medium fritted disk, Gooch crucible 10-15
Whatman 40 filter paper 8
Fine fritted disk, Gooch crucible 4.0-5.5
Whatman GF/D glass microfibre filters 2.7
Very fine fritted disk, Gooch crucibles 2.0-2.5

*Recommended crucible for ADF and NDF analyses.

Filtration difficulties also can be caused by gradual plugging of the fritted disks of crucibles with ash after repeated use. We ash our crucibles as a part of the NDF procedure, but if ashing is not used to determine ash-free fiber values, it should be the first step in cleaning the crucibles after each use. Ash for 5 h at 500-525C. Do not use a higher temperature or the glass will melt or glaze the surface of the fritted disk. Remove residual ash from crucibles with a brush and place them upside down in a sonicating bath containing MICRO cleaning solution and sonicate for 5 minutes (sonication is optional; crucibles also can be cleaned with brush using a detergent solution). Rinse crucibles by pulling water through the fritted disk in the reverse of normal use by connecting a No. 9 rubber stopper with a tube through the middle to a vacuum line containing a trap and applying vacuum. By putting the stopper in the top of the crucible, an air tight fit is achieved for sucking water through the crucible in reverse.

Occasionally the crucibles are cleaned with 6N HCl and/or an alkaline cleaning solution containing 5 g of disodium EDTA, 50 g of trisodium phosphate and 200 g of potassium hydroxide per liter of water. The crucibles should be allowed to soak in either solution for 30 minutes and the alkaline solution should be used with heat. The alkaline treatment can weaken the glass so we use it only on crucibles that do not filter normally. We check the filtration rate of crucibles by measuring the time it takes for 50 ml of water to pass through each crucible without vacuum. It should take approximately 180 seconds. If it takes less than 120 seconds, check the crucible to insure it is not cracked and leaking. If it takes longer than 240 seconds, clean the crucible with acid and measure again. If it is still takes 240 seconds, clean with alkali. If cleaned crucibles take longer than 240 seconds, discard them because they will cause filtration problems.

Drying and Weighing Fibrous Residues. Acetone residues should be removed as completely as possible with vacuum. We recommend that extracted samples be placed near or on the drying oven to evaporate residual acetone before placing them in the oven. We also recommend that all samples be placed in the oven at one time at the end of the day. This prevents moisture from wet samples placed in the oven contaminating samples that have been dried in the oven. Samples should remain in the oven (100-105C) until they achieve a constant dry weight. This normally takes 8 h or overnight drying.

Weighing technique is critical for obtaining dry weights of samples and fibrous residues. We recommend the hot weighing technique because it is more accurate and precise for hygroscopic residues such as dry fiber. The hot weighing technique is faster, requires less handling of the sample and is less prone to the errors associated with desiccator use. If too many samples are placed in the desiccator at one time, if the lid is held open during transfer from the oven or weighing, or if the desiccant is the wrong type or is not changed often, dry weights obtained using a desiccator are incorrect regardless of the oven temperature or drying time. We normally dry residues overnight and weigh them directly out of the oven using a Teflon pad on the balance pan to prevent heat transfer to the balance.

Calculation and Dry Matter Errors. Although it is rare, laboratories have been known to have errors in the equations used to calculate results. The most common source of discrepancies in fiber results among labs is due to differences in dry matter estimates and the variation associated with adjusting fiber values to a dry matter basis. Because dry matter determinations among laboratories are often not in agreement, we routinely compare results among labs on an as-is basis to remove variation due to dry matter adjustments.

Determining Fiber in Difficult-to-Filter Samples

Any sample that takes more than 10 minutes of filtration time under vacuum should be discarded because the results will be inaccurate. Instead, rerun the sample using a modification of the NDF method. Several modifications can be used on any or all samples that are difficult to filter:

  1. Reduce the sample amount to 0.3 g. This will increase the errors associated with weighing and subsampling, but it often is the best approach to use with difficult samples.
  2. Use filter aids. Glass wool (about 0.25 g) or glass microfibre filter mats (Whatman GF/D, 4.25 cm) will keep gelatinous materials and ash or fine residues from plugging the fritted disk of the crucible. The filter mats are expensive, but they sometimes provide the only means of obtaining NDF analyses.
  3. Back-flush the crucible by removing then reinserting it into the crucible holder to force air back through the fritted disk.

High Starch Samples. If filtration is difficult, inject additional standardized amylase solution. Many times this will unplug the fritted disk and allow filtration. Modifications of the original NDF method that use an amylase treatment have solved this source of filtering difficulty in most cases.

High Pectin, Mucilage or Glycoprotein Samples. These samples must be kept hot to filter. Decrease soaking time to the minimum and keep water at boiling temperature. Preheat the crucible by filling it with hot water before beginning to transfer the residue from the beaker. Do not let residues settle in the beaker before transferring to the crucible; instead transfer as quickly as possible. Adding glass wool or glass filter mats to the crucible keep the gelatinous residue from plugging the filter. Adding acetone before the last water wash has been completely filtered (less than 5 ml of water remaining in the crucible) can salvage some samples, but recognize that acetone will precipitate any residual detergent in the residue.

High Fat Samples. Pre-extract samples with acetone to remove some of the lipids before fiber analysis. To do this, weigh the sample into the previously tared crucible that will be used for fiber analysis. Add 30-40 ml of acetone to the crucible and let the sample soak for 5 minutes, stirring occasionally. Remove the acetone with vacuum and repeat the acetone extraction three additional times. After the last extraction, vacuum the sample dry and quantitatively transfer the residue to a Berzelius beaker for fiber analysis. Do fiber analysis in the usual manner and filter the sample into the crucible used initially to extract the lipid. To remove any ND soluble matter that may be retained in the crucible and improve filtration, place it in a heated, covered glass dish with ND solution during the time the sample is being refluxed.

High Ash, Fecal or Digesta Samples. Fecal samples can be especially difficult to filter. It appears that fine material in these samples plugs the pores of the filtering vessel and slows or prevents evacuation. Using microfibre filter mats is usually essential to the determination of NDF in these samples. Filtration also can be enhanced by allowing the sample to settle in the beaker for 60 seconds after it has been removed from the refluxing apparatus and carefully decanting the liquid from the beaker with minimal transfer of particles to the crucible. It helps to slowly transfer the liquid under vacuum in a way that does not cover the entire surface of the filter mat. If the sample begins to plug during the washing step, carefully scrape the surface of the mat to provide a new surface for filtration. Patience and minimum vacuum during the transfer step are important in obtaining accurate results with these samples.

 

<< Appendix E - Taking a Good Forage Sample for Analysis

 

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