Cell wall analysis

Dr Gbola Adesogan

Van Soests Scheme

Based on nutritional availability of forage components I.e. nutritive entities that had the same true digy in all feeds & forages.
Devd for forages, reputed to be variable & difficult to use with other feeds (Mertens, 2001)

Van Soests scheme

Sample
EDTA & Na lauryl sulphate

NFC

NDF Hemicellulose
CTAB + H2SO4

Starches
WSCs

ADF
Kjeldahl

Pectin B-glucans ADIN AIA

Sodium lauryl + ethylenediaminetetraacetic acid (EDTA) Cetyltrimethylammonium (CTAB)
H2SO4/ KMnO4

Ashing

Lignin

Prox. analysis CP EE

Chemical component Protein NPN Lipids Pigments Sugars

Van Soest Analysis

NDS

Organic acids
NFE Pectin Hemicellulose

Alkali soluble lignin

Alkali insoluble lignin CF Fiber bound N

Lignin
NDF ADF

Cellulose
Detergent insoluble minerals
(Fisher et al., 95)

Plant anatomy vs. chemical fractions

(Minson, 1990)

Van Soest (1991)

The NDF and ADF procedures described in the original publications (including Agricultural Handbook 379) are obsolete and of historical interest only.

What does NDF include

Desired contents Structural cell wall components cellulose, hemicellulose, lignin, Pectins (ignored)

Undesired contents Silica, sand Fiber-bound proteins Starch Lipids

Factors affecting NDF values

Processing Subsampling Sample quantity Grinding Drying

Method
Reagent quantity Boiling duration

Reflux time & temperature

Filtration method and vessel Soaking Weighing method

NDF procedural differences

(Van Soest 91)

Pre or post treatments

Lipid removal >10% of lipid can be problematic Acetone dissolves fats Ethanol also used to dissolve lipids Protein removal For high protein (>30%) feeds Proteases Sulfite Originally included to NDF bound nitrogen e.g. keratin Still useful for high protein forages Solubilizes lignin cant use in sequential analyses leading to lignin determination / for in vitro digy.

Pre / post treatments

Ash Express results on OM (ash free) basis to eliminate soil contamination
Decalin Originally included to prevent foaming Increases fiber yield

Starch contamination
Can overestimate NDF Starch removal simplifies filtration Amylase treatment Which amylase ? Not Bacillus subtilis May contain undesired enzymes - hemicellulase, glucanase & protease activity High temperature treatments reduce undesired activities Enzyme activity variations For concs, combine amylase pretreatment with 2 Ethoxyethanol effective; but is a health risk, Replace with triethylene glycol Urea treatment

Effect of amylase source on NDF

ND- S2 =Bacillus subtilis; ND-T3 heat stable bacteria

(Van Soest, 1991)

Limitations of NDF method

Not nutrient-based Doesnt give chemically/anatomically pure fractions Fiber itself is not chemically, physically or nutritionally uniform Doesnt differentiate between polysaccharides sufficiently Ignores pectin & glucans Pectins unique fiber which is Not digested by mammalian enzymes Rapidly fermented by rumen microbes

ADF
Though often used to predict digestibilty Van Soest claims No valid theoretical basis to link ADF to digy. ADF is a preparative residue for isolating: Cellulose Desirables Lignin Maillard products Silica Undesirables AIA ADIN

Factors affecting ADF

Acid strength Boiling time

Contaminants

(McLeod & Minson 72)

ADF contaminants
Sulphuric acid removes most of the digestible fiber

CTAB removes some protein leaves fiber-bound protein ADF residue contains pectin & hemicellulose except if determined by sequence after NDF extraction
Express on OM basis to eliminate AIA Determine ADIN to account for indigestible N Dont use asbestos packed crucibles for filtering

MADF
UK replacement for ADF Longer boiling time, Stronger acid Gave a more accurate digy. prediction than ADF Shouldnt be used to assay ADIN / heat damaged protein since MADF sample must be oven dried

ANKOM NDF
Ideal for difficult to filter samples E.g. Silage or soil contaminated samples Precise Eliminates most elements of Technician variability. Consistent with conventional and alternative method results. Efficient Reduces labor Processes up to 24 samples at a time. Safety Eliminates handling of Hot chemicals. Space Saver Instrument requires little space for operation

Lignin
What is lignin
Non- CHO substance that resists digestion not a well defined, individual compound Complex, cross-linked polymer containing phenylpranoid units derived from Coumaryl alcohol Coniferyl alcohol Sinapyl alcolhol (McDonald et al., 95) Functions In plants structural In ruminants decreases energy density & digestibility

Methods of lignin analysis

Most based on lignin insolubility in 72% H2SO4 (I.e Klason lignin) Overestimates lignin due to co-precipitation of Protein Malliard products Cutin Tannins Protein contamination can be reduced with Protease pretreatment ADF pretreatment

Lignin methods
ADF lignin ADF pretreatment followed by sulfuric acid or permanganate solution Dilute acid (1M H2SO4) at 100oC Followed by Conc acid (12 M H2SO4 at 25oC) Residue is lignin Klason lignin Pretreatment with ethanol, amylase & amyloglucosidase Acid hydrolysis conc (12M H2SO4) at 39oC followed by (0.4M H2SO4) Residue is lignin

Lignin methods
Gravimetric methods Lignin is left as the residue after the digest ADL underestimates lignin due to lignin solubilization in the acid
Difference methods Lignin is solubilized / oxidized and determined by difference Can use chlorite, permanganate etc. Absorbance method Lignin is solubilized (e.g. with acetyl bromide) and then determined spectophotometrically

Lignin methods
Saponification method Lignin is determined by cleavage of the ester linkages in lignin Ball milling
Pulverized sample amongst ball bearings for long periods of time. A portion of the lignin can then be extracted with certain solvents.

Lignin methods
Pyrolysis mass spectroscopy Pyrolysis thermal degradation of sample in an inert atmosphere or a vacuum. mass spectrometer used to separate the components of the pyrolysate on the basis of their mass-to-charge ratio Calorimetry Based on compairing the actual gross energy of the sample to a calculated GE based on energy values of the chemical components in the samples Calculated GE = (Protein x 5700kcal/kg) + (Carbohydrate x 4000 kcal/kg) + (lipid x 9500 kcal/kg) + (lignin x 8000 kcal/kg)

% GE recovered
Sample Actual GE Kcal/kg 4493 4463 4497 4182 Calculated GE from ADL 80.8 76.2 78.4 73.8 Calculated GE from Klason lignin 97.1 87.6 93.7 96.7 (Jung & Vagel, 1996)

Alfalfa Clover Corn silage Oat straw

Lignin analysis methods

(Cherney, 2000)

Problems with lignin assays

No standardized method Reasons Complex unknown structure No standardized method Variation in lignin content with forages Leading to poor predictions of digy. No current method accurately measures pure lignin (Giger 1985) Better digestibility predictions from lignin expressed on a fiber basis Lignin/NDF

References
Varel VH, Weimer PJ, et al. Accuracy of Klason lignin and acid detergent lignin methods as assessed by bomb calorimetry J AGR FOOD CHEM 47 (5): 2005-2008 MAY 1999 Abrams SM Sources of error in predicting digestible dry-matter from the acid-detergent fiber content of forages ANIM FEED SCI TECH 21 (2-4): 205-208 OCT 1988 D.J.R. Cherney Characterization of Forages by Chemical Analysis. Forage Evaluation in ruminant Nutrition. Eds Givens, Owens & Ohmed. CABI Van Soest, P.J., Robertson, J.D. and Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber and non-starch polysaccharide in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597. Jung HJG, Mertens, D R and Payne, A J. 1997. Correlation of acid detergent lignin and klason lignin with digestibility of forage dry matter and neutral detergent fiber. J Dairy Sci. 80: 1622-1628 http://www.cabi-publishing.org/Bookshop/Readingroom/browseA-Z.asp