ADL Van Soest, insoluble lignin in H2S04: what is the most appropriate method for analyzing the ligneous fraction of raw materials and rabbit feeds?

Fibers, particularly the ligneous fraction, are a key component of a rabbit’s diet. With this in mind, rabbit feed formulators must absolutely be provided with an accurate determination of the fiber content of raw materials. Our studies showed that Van Soest’s ADL analysis leads to inaccurately high readings of the lignin value for some raw materials, especially if these are high in polyphenols, such as grape byproducts. Eventually this overcalculation is likely to induce a lignin deficit in rabbit feed.

Fibers, lignin in particular, play an essential role for ensuring rabbits’ digestive health. Therefore the share of lignin in raw materials and rabbit feed must absolutely be determined accurately so that the specific requirements of rabbits are met. Several methods are used for determining the concentration of lignin. The most commonly applied analysis are Van Soest’s and that of the insoluble lignin in H2SO4. Therefore which one is the best for determining the lignin content of raw materials intended for rabbit feeds?

Significant deviations between Van Soest’s technique and the direct method in H2SO4

A study run by TECHNA FRANCE NUTRITION showed that the Van Soest’s ADL method of analysis and that of the lignin H2SO4 came up with different results when these analyses were applied to specific raw materials. In grapes byproducts, the ADL value (Acid Detergent Lignin) obtained with Van Soest’s method was much higher (up to more than 7.7 pts for grape byproducts) than the lignin value resulting from H2SO4 analysis. A slightly lower deviation was observed when analyzing sunflower hulls.

In order to determine the most accurate approach, we proceeded to examine the composition of a fibrous mix of raw materials - the Lapilest®. Each of the raw materials composing the Lapilest® was thoroughly analyzed. The weighted sum of each raw material was compared against the analytical value of the fibrous mix. While the best additivity was achieved when applying the H2SO4 method, the ADL value of the mixture turned out to be higher than what was initially expected.

This gap clearly showed an over calculation of the lignin content with Van Soest’s method, while the H2SO4 method reflected faithfully the unitary values of each raw material. When analyzing specific raw materials, we ended up having an over calculation of the lignin levels with Van Soest’s method, such as on grape byproducts. This result was also demonstrated on the carob. The conclusion is that a rabbit feed whose formulation is based on Van Soest’s ADL method may end up presenting a lignin deficit. This deficit might have heavy consequences on rabbits’ health (digestive issues, deaths...).

The polyphenol content is the key to the deviations observed between the two methods

Carob and grape byproducts are notable for their significant content in phenolic compounds. The compound analysis showed that these raw materials are particularly high in polyphenols (3380μg / g for the grape pulp and 5208μg / g for grape seed) with a significant share of flavonoids (87.6% and 92.9%. The flavonoid subfamily of compounds also contains tannins.

Sunflower hulls contain an intermediate level of polyphenols (1042 mg / g) yet these polyphenols do not belong to the flavonoid sub-category. Apple pomace also contains smaller amounts of polyphenols (602 mg / g) while the beet pulp is devoid of them.

Bibliographic data show that in the presence of tannins, the stage of neutral detergent attack of Van Soest’s protocol results in the creation of protein-tannin complexes. In addition, the various stages of high temperature drying (3 times for 3 hours at 103 ° C) increase the polymerization of tannins and their complexation with other cellular components (fibers, proteins). Yet components such as fibers or proteins often cannot be completely solubilized during acid attacks (acid detergent and H2SO4). Therefore the ADL residue obtained by Van Soest’s method ended up containing not only lignin, but also polymerized tannins. This accounts for the inaccurate high readings of lignin.

What are the teachings of these findings on lignin for our rabbit feed formulation?

With these teachings in mind, our rabbit experts have resolved to further improve the accuracy of lignin measurement in rabbit feed. From now on, our formulas will take account of two different criteria (or nutrients):

• the ADL, based on the analysis results of the Van Soest’s method, or on a specific calculation resulting from our experiments,
• the lignin, stemming from our H2SO4 analysis results or from our own equation improved following these findings,

However, our nutritional specifications for feed formulation will still take account of the H2SO4 lignin criterion.

These experiments shed light on the differences between two analysis of lignin content on specific raw materials and a mix of these raw materials. These differences stem from the formation of complexes with polyphenols (tannins). In light of these findings, our specialists recommend to preferably use the insoluble lignin in H2SO4 method for determining the ligneous fraction in raw materials (or rabbit feed) that have a high polyphenol content. Still, we consider that Van Soest’s ADL method is reliable when applied to slightly ligneous raw materials and to raw material with a small concentration of polyphenol. For more information on methods relating to the lignin content, please contact us!