|Livestock Research for Rural Development 9 (1) 1997||
Citation of this paper
Use of the nylon bag technique for protein and energy evaluation and for rumen environment studies in ruminants
E R ěrskov and W J Shand
International Feed Resource Unit, The Rowett Research Institute,
Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK
The nylon bag technique is a very robust and powerful tool with which to study several aspects of nutrition in ruminants. It is particularly useful in describing degradation characteristics of protein and roughages and also for rumen environment studies. For each purpose a slightly different approach has to be used. This article explains how this is best achieved.
Key words: Rumen, nylon bag, digestibility, degradation characteristics, gas production
We have been asked to write a small article relating to the nylon bag technique and interpretations of results. Complications sometimes occur as the technique is used for different purposes, namely protein evaluation, roughage evaluation and evaluation of rumen environments. We would like to treat each one in turn.
The dynamic nylon bag procedure was first developed to estimate degradability of protein and for this a formula was developed ( ěrskov and McDonald 1979)
p = a + b (1 - e-ct) (1)
where Ap@ is degradation at time At@ and Aa@,@b@ and Ac@ are constants and Ae@ is the basis of the natural logarithm. The equation was particularly useful since the constants had a biological meaning, Aa@ being the intercept or the immediately soluble fraction, Ab@ the insoluble but rumen degradable fraction, and Ac@ the rate at which the insoluble rumen degradable fraction is degraded. It follows that 100 - (a + b) is the total rumen undegraded fraction. Part of this fraction, depending on the protein source, will be degradable in the small intestine.
Figure 1: Loss of protein from nylon bags at intervals during 72 hours
Let us construct a situation (Figure 1) in which bags are withdrawn at intervals of 2, 4, 8, 16, 24, 48 and 72 hours and the protein disappearance values are 41.5, 49.6, 59.0, 69.5, 74.8, 75.6 and 75.8 then Aa@ = 32.1, Ab@ = 44.0 and Ac@ would be 0.1229 respectively and the residual standard deviation, 0.71. In other words the data fitted the formula well.
The next problem was that since protein supplements consist of small particles which are small enough to leave the rumen, these particles could escape the rumen so that two possibilities existed: an insoluble particle entering the rumen could be degraded, depending on the degradation rate Ac@ or it could flow out, depending on the outflow rate, here denoted as Ak@. In order to describe this we coined the word effective degradability AP@ representing that which was actually degraded in the rumen. This was expressed by:
P = a + b (c/(c + k)) (2)
It can be seen that as Ak@ or outflow rate increases AP@ will, of course, decrease. Let us for example use three values for Ak@ in the equation above namely 0.02, 0.05 and 0.08 which gives effective degradabilities of 69.9, 63.4 and 58.8, respectively.
It must be remembered that this formula should only be used when the feed consists of small particles. For long forage particles it is not applicable, since the time taken to reduce long to small particles by chewing, and microbial disintegration, is not taken into account and this is likely to vary between feeds. For protein supplements therefore it is quite simply the use of formulae 1 and 2.
For several years the 48 hour degradability was used as an approximation to in vivo digestibility and for this it is still used. However, it has since been realised that some of the plant factors affecting consumption of roughages could also be identified by the dynamic nylon bag approach, namely the soluble, the insoluble and fermentable fractions, and the rate at which the insoluble material is fermented. However, here there is a complication which is not so apparent for protein supplements, namely a lag phase when microbes become attached to the fibrous material during which time there is no net disappearance of substrate. In fact, there may even be an increase in weight during the first 2-4 hours. On the other hand the soluble material will disappear rapidly.
There are different ways of dealing with this. In our laboratory we have chosen the following approach. Due to the lag phase, during which time there is no net disappearance of substrate, the Aa@ value in the equation (ie: the extrapolated value) could be negative. As an example, take the situation where samples are removed at 8, 24, 48, 72 and 96 hours after incubation and the typical disappearance values (%) for a roughage are 17.0, 42.1, 51.6, 55.3 and 56.2.
Applying the formula p = a + b (1 - e-ct) gives Aa@ = -7.9, Ab@ = 63.8, Ac@ = 0.0622. RSD = 0.94. Obviously, the solubility cannot be negative. This predicted negative value is due to the lag phase.
Solubility can be determined by different methods such as washing nylon bags, containing the substrate, without incubation in the rumen. This value is called AA@, the value for solubility determined in the laboratory. It is clear that (a + b) represents the asymptote (ie: the maximum potentially fementable material). It is then easy to appreciate that the asymptote (a + b), less the solubility AA@, represents the insoluble but fermentable material. This is called AB@ to distinguish it from Ab@ which is from the mathematical expression. Ac@ is the same as the rate constant generated from the equation. For roughage evaluation therefore the situation is:
A = solubility
B = (a + b) insoluble but fermentable
c = rate constant
From the previous example AA@ was 10.6% thus AB@ = (-7.9 + 63.8) -10.6 = 45.3 and Ac@ is 0.0622. In some trials, multiple regressions using AA@, AB@ and Ac@ have been closely related to feed intake, digestibility and animal performance but more information is required from trials in which both feed intake and degradation characteristics are determined for less conventional feeds such as the leaves from trees and shrubs.
Should incubation periods be different for roughages? Ideally the first bag should be withdrawn only after the lag phase is completed. It is advisable not to withdraw any bags from the rumen before 8 hours. It is essential also that the asymptote is well described (ie: the differences between the losses for the last incubation periods must be small, no more than about 5% of the final value), otherwise the asymptote will be extremely inaccurate and sometimes incubation periods of up to 120 hr are required.
In order to rank the intake potential of the feeds an index value has been derived from regression equations using AA@, AB@ and Ac@ as independent variables to predict intake. The index derived from a number of roughages was:
Index value = A + 0.4*B + 200*c.
These coefficients will be different for different groups of feeds. For cattle in Europe it seems that an index value of about 30 is needed to achieve maintenance energy intake.
The index value of the feed described previously is:
10.6 + 0.4*45.3 + 200*0.0622 = 41.1.
Evaluation of rumen environment
In the above examples it was assumed that the rumen environment was optimal. The substrate is varied and it is assumed that there is an optimal rumen environment. The nylon bag method can also be used to determine the optimal concentration of NH3, S or the optimal pH. In this case the approach is to vary the rumen environment but keep the incubated substrate constant. As standard substrates one can use ground straw, soya bean hulls or other uniform cellulosic substrate. Sometimes it is useful to wash the soluble material out of the substrate as this material will disappear, regardless of the rumen environment.
It must be understood that the rumen environment will not affect AA@ nor the asymptote but it will affect the time taken to reach the asymptote. In other words it is the Ac@ value that will be sensitive to the rumen environment.
The nylon bag technique is a very powerful and robust tool but it is important to understand the purpose for which it is to be used: for evaluating protein supplements or roughages, or for evaluating dietary effects on the rumen environment.
The gas production method
A complementary tool to the nylon bag method is the gas evaluation technique in which substrate is incubated in syringes and the gas produced is measured at intervals of time. The same equation as above [p = a + b (l - e-ct)] can be used and the gas produced at intervals can be used to evaluate feeds. Many comparisons of the two techniques have been published (see Blummel and rskov 1993). The gas production method can also be used to evaluate antinutritive factors targetting microbes, in so far that complexing agents like polyethylene glycol (PEG) can be added to the substrate and the gas production with and without complexing agents can be used to measure the presence of microbial anti-nutritive factors.
Blummel M and ěrskov E R 1993 Comparison of in vitro gas production and nylon bag degradability of roughages in predicting feed intake in cattle. Animal Feed Science and Technology, 40: 109-119.
Makkar H P S, Blummel M and Becker K 1995 Formation of complexes between PVP or PEG and tannins, and their implication in gas production and true digestibility in in vitro techniques. British Journal of Nutrition, 79: 897-913.
ěrskov E R and McDonald I 1979 The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. Journal of Agricultural Science, Cambridge. 92: 499-503.
Received 9 January 1997