• 4.

Evaluation of feedstuff (digestibility trials)

• Chemical analysis indicates the potential value of a feed for supplying a particular
nutrient, but the actual value of a feed to the animal can be arrived only after
making allowance for the inevitable losses that occur during digestion, absorption
and metabolism. The first tax imposed on the feed is that represented by the part
of it which is not absorbed and is therefore excreted in the feces.
• Digestibility refers to the disappearance of feed or food from the gastrointestinal
tract. In other words, digestibility is the degree of degradation of feedstuffs in the
gut. Digestibility coefficient is therefore, the proportion of which is not excreted
in the feces and which is therefore, assumed to be absorbed by the animal thus
available for metabolism. Therefore, digestibility is an essential feature of
feedstuff evaluation. As it has been said digestibility measures the amount of
nutrients in a feed that is digested and absorbed and thus available for
metabolism. For instance, if row fish meal contains 90% CP but the digestibility of
the protein is only 10%, this means the potential usable CP content is only 9%.
1
 Factors that may affect digestibility
• Digestibility results of the same feed may be variable because of different factors.
These include:
1. Level of feed intake
• An increase in the quantity of feed eaten by an animal generally causes a faster
rate of passage of digesta, meaning the feed will be exposed to the action of
digestive enzymes for a shorter period, and there may be a reduction in its
digestibility. This has higher effect for the slowly digestible components of feeds
namely cell wall constituents.
• Levels of feed intake are often expressed as multiples of maintenance
requirement (maintenance requirement defined as unity). Where an animal is
given above maintenance requirement the animal is not efficient in its
digestibility because the animal has plenty and digestive system digests enough.
Therefore, we have to keep an animal not over fed when we use them for
digestibility trial.
2
2. frequency of feeding
• This is related to the level of feed intake. If an animal is fed 3 meal systems and an
animal fed continuously, the digestion will be lower in the latter group. Because
when an animal is fed 2-3 times a day, this will give more time to be attacked by
enzymes of the microbes and host resulting to more efficient digestion than the
animal fed continuously.
3. Animal factor
• Feed offered to different animals will not always be digested to the same extent.
Therefore, species of consumers will affect digestibility. Feeds low in fiber are
equally well digested by ruminants and non ruminants but more fibrous feeds are
better digested by ruminants.
• Difference in digestibility ability between sheep and cattle is small and is of little
importance with most diets. However, high digestible feeds like cereal grains tend
to be more efficiently digested by sheep and low quality roughage tend to be
better digested by cattle.
3
• A major animal factor influencing efficiency of roughage utilization is the
rumination rate. Rumination is important in helping to degrade fibrous material
into smaller particle sizes, facilitating their digestion by microbes. The maximum
time spent in rumination is 8 to 9 hours per day. So the more roughage that can
be ruminated during the 8 to 9 hours per day, the greater the efficiency of
digestion, roughage intake and productivity.
• Cattle are more efficient in rumination than sheep and goats are intermediate
and cattle are better than sheep in utilizing poor quality roughages. Calves
ruminate less efficiently than mature bovines. Thus younger animals should be
fed better quality hay than mature once.

4
4. Digestive disturbance
• If an animal has got digestive disturbance, digestibility will not be efficient. Animal of
normal digestive tract and that with digestive disturbance if given same feed,
digestibility will not be the same.
5. Nutrient deficiency
• If an animal is deficient in certain nutrients, it will not be in a position to efficiently
digest the feed eaten. For instance if there is a protein deficiency since most enzymes
and hormones are proteins, the lack of it will result to lack of enough enzymes and
hence digestibility will be affected.
6. Feed composition
• The digestibility of a feed is closely related to its chemical composition. The fiber
fraction of a feed has the greatest influence on its digestibility, and both the amount
and the chemical composition of the fiber are important.
• Feeds having low nitrogen may result to a negative digestibility coefficient for apparent
digestibility of proteins because of a higher endogenous nitrogen loss for the wall of
the gut compared to the exogenous origin. This also holds true for ether extract.
5
7. Feed processing or preparation of feeds
• If we grind alfalfa coarsely, this may help the animal to digest the feed favorably
as compared to the untreated alfalfa which may need much more time for
digestion. On the other hand if the alfalfa is ground finely, the rate of passage of
the feed across the gut will be high without being much exposed to enzymatic
action. So processing will affect digestibility both in a positive and a negative
manner.
• The other processing is heat treatment. It helps in improving nutritive value and
digestion if used properly. Over heating on the other hand may cause less
digestibility. Other treatment is alkali treatment which improve digestibility of
lignified feeds.

6
8. Ration composition (effects of combining different feeds)
• The digestibility of a feed is influenced not only by its composition but also by the
composition of the other feeds consumed with it. We use individual feeds for
digestibility trial especially for forages. But as concentrates can not be fed to the
animals alone as the system of ruminants not adapted to concentrate alone, they
are combined with other ingredients.
• In digestibility trial we can not get an added effect. Certain feeds may have
nutrients which has negative effect to digestibility coefficient than the lower. So
combining feed ingredients may alter digestibility of feeds of unknown
digestibility coefficient. When feeds are combined, digestibility coefficient of
individual feeds will be lost.

7
• Fermentation of soluble carbohydrates of the concentrate may modify the rumen
environment and depress the digestibility of the cellulose and other fiber components of
the roughage. Too little nitrogen also decreases fiber fermentation. If digestibility of the
roughage is 0.6 and concentrate 0.8, digestibility of the mixture is not necessarily 0.7.
• The population of rumen microbes reflects the nature of the diet consumed. Roughage
diets are high in cellulose, low in starch, and intermediate in soluble sugars, thus
supporting a microbial a microbial flora of mainly cellulolytic and saccharolytic (sugar
digesting) bacteria. These organisms produce acetate as their main fermentation end
product.
• With high starch diets, amylolytic bacteria predominate, They ferment starch, sugars and
hemicellulose to propionate. The rate of digestion of cellulose is much slower than for
soluble carbohydrates. The rumen pH is lower when high concentrate diets are fed than
when high roughage diets are fed. The reason for this includes:
Decreased rumination time,
Reduced salivation,
High rate of propionate production, and
High lactic acid production 8
• Measurement of digestibility
• In a digestibility trial the amount of feed under investigation that is given to the
animal shall be known and the output of feces shall be measured. In such trials
more than one animal is used because animals even of the same species, age and
sex differ in their digestive ability and secondly replication allows more
opportunity for detecting experimental error.
• In trial with mammals male animals are preferred than females. This is because it
is easier to collect feces and urine separately with males. The animal for
digestibility trial has to be docile and in good health.
• For small animals like sheep, the animal may be confined in metabolic cages,
which facilitate the separation of feces and urine by an arrangement of sieves.
For large animals such as cattle, it is easier to fit them with harness and feces
collection bags made up of rubber or a similar impervious material.

9
• For poultry, the determination of digestibility is complicated by the fact that feces and
urine are voided from a single orifice, the cloaca. The compounds present in the urine
are nitrogenous and can be separated chemically with the assumption that most urine
nitrogen is in the form of uric acid and fecal nitrogen is in the form of true protein. The
other possibility is to alter the fowl’s anatomy by surgery so that urine and feces are
voided separately.
• The feed required for digestibility trial should be thoroughly mixed in advance to get or
ensure uniform composition. It is then given to the animal for at least a week before
starting fecal collection in order to accustom the animal to the diet and cage, and to
clear from the tract the residue of previous feeds. This is called preliminary period.
• Preliminary period: the animal which is exposed to the digestibility trial needs time to
be acclimatized with the feed and cage the animal kept in. The period could be 7 to 20
days depending to the type of feed we want to test. If the feed under consideration is
new to the animal, which may alter the physiology of the gut like concentrates to
ruminants, the animal should be given a relatively longer time so that it can get
accustomed to the diet and eat much of it. If the feed is grass or feed that the animal is
used to eat, it can be a shorter duration.
10
• With regard to the cage, if the animal never had been confined in cage before, time has
to be given to the animal to adapt for the cage before commencement of data
collection. Otherwise the animal will get nervous and not consume much which will
affect digestibility value.
• The preliminary period is followed by a period in which feed intake and fecal output is
recorded. This experimental period is usually 5-14 days long with longer periods giving
greater accuracy.
• Arbitrary time gap of 24 to 48 hours is normally allowed for the passage of feed
residues. Therefore, the measurement of fecal output begins 1 to 2 days after that of
feed intake, and continues for the same period after measurement of feed intake has
ended.
• In digestibility trial meals should be given at the same time each day and the amount of
feed eaten should not vary from day to day. When intake is irregular, for example, if the
last meal of the experimental period is usually large, the subsequent increase in fecal
output may be delayed until after the end of the fecal collection. In this situation the
output of the feces resulting from the measured intake will be underestimated and
digestibility overestimated. The trial is complete by analyzing samples of the feed used
11
and feces collected for different nutrients for digestibility determination of nutrients.
• Methods of digestibility determination
• After undertaking chemical analysis, we undertake digestibility trial to see how
much of the ingredients are degraded in the digestive system. There are different
methods of estimating digestibility. This include
A. In Vivo/Conventional digestibility
B. Laboratory method (In Vitro)
C. Difference method
D. Indicator method

12
• 4.1. In Vivo/Conventional digestibility
• In vivo Describing biological processes as they are observed to occur in their
natural environment, i.e. within living organisms. Thus, In Vivo (Conventional
digestibility) is a direct measurement that involves keeping an animal in a
metabolic crate and measuring the feed intake and the fecal output. The feed and
feces are analyzed for nutrients of interest to determine nutrient digestibility. The
difference between the amount of nutrients consumed and excreted in the feces
is the amount digested and absorbed. Therefore, this requires quantification of
feed consumed and total feces excreted. For ruminants sheep is mostly used.
• 
• % Digestibility = DM in feed – DM in feces x 100
• DM in feed
• 

13
• Example: A cow consumes 10 kg of hay which has 90% DM, and excreted 3kg of
DM in the feces. What is the digestibility of the hay DM?
• 
• % Digestibility = DM in feed – DM in feces x 100
• DM in feed
• = (9 – 3) x 100
• 9
• = 66.6%

14
• One may undertake nutrient digestibility to quantify what % of protein, ether extract, or
other nutrients digested in the conventional method. In this case, we use the following
formula:
• % Digestibility = Nutrient intake – Nutrient in feces x 100
• Nutrient intake
• Example: A pig is housed in a metabolism cage where feed intake and fecal output is
measured. It consumes 20 kg of the test diet, and excreted 10 kg of feces. The percentage
of CP in the feed is 15 and of the feces is 7. What is the protein digestibility of the diet?
• Protein intake = 20 kg x 0.15 = 3 kg
• Fecal protein = 10 kg x 0.07 = 0.7 kg
• % Digestibility = Nutrient intake – Nutrient in feces x 100
• Nutrient intake
• % Digestibility = 3 – 0.7 x 100
• 3
• = 76.6%
15
• Shortcoming of conventional method
It is laborious and time consuming.
In conventional method, we get the apparent digestibility. The ingredients or the
composition of the feces is one of the weaknesses of this method. Not all feces are
actual undigested feed residues. With in the feces there are nutrients coming from the
wall of the digestive tract. In the due course of metabolic reactions minerals, enzymes
and dead cells from the digestive tract is contaminated with the feces but when we
analyze, we consider as if all theses materials are part of the feed consumed. That will
underestimate digestibility because of the problem of quantifying the undigested
material.
• When one estimates digestibility of protein free feeds, since feces contain protein that
originate from the animal (metabolic fecal nitrogen), negative digestibility results may
be obtained. Therefore, to quantify true digestibility one has to be able to quantify feces
that come from the feed and the animal. This also holds true for ether extract. Ether
extract in ruminants is of two origins. Microorganisms produce fat which may mostly
appear in the feces and affect our estimate of digestibility, that is why we say apparent
digestibility (the nutrient that appear in the feces is from two sources but hard to
differentiate). 16
• On the other hand the methane arising from the fermentation of carbohydrates is
lost by eructation and not absorbed. This loss leads to overestimation of the
digestible carbohydrates and digestible energy content of ruminant feeds, which
will overestimate digestibility. Therefore, values called apparent digestibility not
true digestibility. The fraction of the feces that is originated from the feed and
from the animal is in most cases indistinguishable from one another.
• 4.2. In Vitro digestibility (Laboratory method)
• Since digestibility trials are laborious and expensive to carry out, numerous
attempts have been made to determine the digestibility of foods by reproducing
in the laboratory the reactions that take place in the alimentary tract of the
animal. Digestion in non-ruminants is not easily simulated in its entirety, but the
digestibility of food protein may be determined from its susceptibility to attack in
vitro by pepsin and hydrochloric acid. It is also possible to collect digestive tract
secretions via cannulae and to use them to digest foods in vitro.

17
• The digestibility of feeds for ruminants can be measured quite accurately in the
laboratory by treating them first with rumen liquor and then with pepsin. During
the first stage of this so called two-stage in vitro method a finely ground sample
of the feed is incubated for 48 hours with buffered rumen liquor in a tube under
anaerobic conditions. This first stage involves test tube containing buffer solution,
the rumen microbes and test forage incubated at a body temperature under
anaerobic condition. The buffer solution represents the artificial saliva and buffers
the acid produced during fermentation.
• The rumen microbes are obtained by collecting rumen fluid from a fistulated
animals and straining the fluid to remove particles. The test forage is added
individually in weighed amounts (0.5 g) to tubes, which are gassed with carbon
dioxide to create anaerobic condition. The tubes are placed in a water bath and
are incubated at 37 0C. In the second stage the bacteria are killed by acidifying
with hydrochloric acid to pH 2 and are then digested by incubating them with
pepsin for a further 48 hours.

18
• The insoluble residue is then filtered off, dried and ignited and its organic matter
is subtracted from that present in the feed to provide an estimate of digestible
organic matter. By measuring the amount of DM or other nutrients of interest in
the forage before and after incubation, the digestibility of each fraction can be
determined.
• IVDMD % = Initial dry sample wt – (Residue – Blank) x 100
• Initial dry sample wt
• The feed or forage sample is incubated with rumen fluid and a buffer solution
(artificial saliva) under anaerobic condition, at body temperature (37 0C). Such a
system stimulates the digestive activity of the rumen and is much simpler and less
expensive to perform than in vitro digestibility trial with animals.

19
• In vitro digestibility techniques provide a quick, inexpensive and precise prediction
of in vivo or conventionally determined digestibility in ruminants. It has an
advantage in that a lot of sample can be easily tested using only a few grams of
feed samples whereas metabolism trials are lengthy, expensive and require large
number of feeds and many animals. There are however different variables that
affect the in vitro method. The four major once are:
1. Variation in the microbial population
2. Variation due to different storage, grinding and processing techniques in sample
preparation
3. Differences attributable to the fermentation medium
4. Length of fermentation and laboratory errors
• Therefore one has to try to control and standardize the above mentioned
parameters to get a reasonable estimate of digestibility despite all the above
factors affecting in vitro value, the technique is still the best means of laboratory
evaluation of digestibility available today.
20
• 4.3. In Sacco / nylon bag techniques digestibility
• The other laboratory method is the nylon bag or In Sacco or in situ method of
digestibility determination. In this method, instead of doing laboratory estimation
of digestibility, fistulated animals are used. Feed samples 2-3 g will be placed in
small bags made up of permeable synthetic fabrics of standard pore size 50
microns or less which will be inserted into the rumen through the cannula and
incubated there for 48 – 72 hours. After each bag will be withdrawn, washed and
dried to determine the quantity of feed DM remaining as undigested material.
Digestibility will then be determined by difference.
• In the nylon bag method there are problems in its use arising particularly from
the need to select an appropriate period of incubation. But it has a special
application in the assessment of protein breakdown and rate of digestion in the
rumen.
• The problem of the laboratory technique of digestibility estimation is that, the
value tells us the rumen digestibility since we simulate what is happening in the
rumen. But digestion occurs from ingestion to excretion. 21
• There is also a possibility to estimate the digestibility of feeds in successive sections of the digestive tract by
exteriorizing the tract at selected points. Fistula or cannula could be fitted at different points and the fate of
nutrients can be determined by collecting digesta from successive portions of the gut.
• Digestibility determination is generally laborious, time consuming and expensive. Therefore, instead of
running the actual digestibility trial, estimation of digestibility was derived from results of chemical analysis
of Van soest.
• %EDDM = 0.98S + WDC – M
• Where,
• EDDM Estimated digestibility of dry matter
• S Cellular contents
• W % cell wall constituents
• DC Estimated digestibility coefficient
• M Metabolic fecal matter, average value for a ruminant is 12.9%.
• All values must be expressed as percentage. If there is silica, it will be counted as lignin.
• Digestibility coefficient (DC) can be estimated using the following formula.
• DC = 108.8 – 96.6 log10 ((l/ADF) x 100)
• DC = 147.3 – 78.9 log10 ((l/ADF) x 100)
• The two formulas are used for DC estimation. The first formula is for Permanganate lignin while the second if
for 72% H2SO4, as there is difference in lignin quantification using the two methods. 22
• 4.4. Indicator/marker techniques of digestibility
• Sometimes it becomes impractical to measure directly either feed intake or fecal
output, or both. For instance when animals are fed in a group, it is impractical to
measure the intake of each individual. Digestibility is however can still be
measured if the food contains certain substances which are known to be
completely indigestible. These substances are called indicators. There are two
types of indicators. These are:
• Internal indicators
• These are indicators found with in the feed. Such indicators are indigestible
components of the feed or are natural constituents of a feed. Lignin is the
common internal indicator. Acid insoluble ash (silica) and indigestible ADF could
also be used as internal indicator.

23
• External indicators
• These are indicators that we add to the feed. These may be synthetic products
such as chromic oxide (Cr2O3) which is mostly used one and ferric oxide. For non
ruminants titanium dioxide may be added to feeds as an indicator. These
products are insoluble and hence indigestible and are unlikely to be present as a
major constituent of feeds.
• If the concentration of the indicator in the feed and small sample of feces of each
animal is measured, the ratio between the concentrations gives an estimate of
the digestibility of the feed.

24
• Internal or external indicators may be used. Internal indicators are natural
constituents of the food such as lignin, acid-indigestible fiber or acid-insoluble ash
(mainly silica). More recently, the long-chain hydrocarbons (n-alkanes, C25–C35)
found in the waxy cuticle of leaves have been used as internal indicators,
especially in grazing studies. External indicators are substances that are added to
foods. Chromic oxide (Cr2O3) is perhaps the most common external indicator as it
is very insoluble and hence indigestible; moreover, chromium (Cr) is not present
as a natural constituent of most foods. In non-ruminant nutrition, titanium oxide
(Ti2O3) is often used as an external indicator.
• External indicators such as chromic oxide may also be used to estimate fecal
output rather than digestibility. In this application, the indicator is normally given
for 10–15 days in fixed amounts (e.g. administered in a gelatin capsule) and once
its excretion is assumed to have stabilized its concentration in fecal samples is
determined. Fecal dry matter output (kg/day) is then calculated as: Indicator
dose (g/day)/indicator in feces (g/kg DM)
25
• For example, if an animal is given 10 g of chromic oxide per day and the
concentration of indicator in the feces is 4 g/kg DM, then feces output would be
calculated as 10/4 = 2.5 kg DM/day. If food intake is known, then dry matter
digestibility could be calculated as (dry matter intake - fecal DM output)/DM
intake. Alternatively, if DM digestibility is known, then dry matter intake could be
calculated as fecal DM output/DM digestibility. The n-alkane technique is very
useful in this context. As plants contain mainly odd-chain n-alkanes in their waxy
cuticle, even-chain (C32) n-alkanes can be used as an external indicator to
determine fecal output. At the same time, the odd-chain n-alkanes (C35) can be
used to estimate diet digestibility. Dry matter intake can then be estimated in
group-fed or grazing animals.

26
• Measuring the digestibility of herbage eaten by grazing animals presents a
particular problem. In theory, internal indicators such as lignin can be used to
estimate herbage digestibility. However, in practice, this application of the indicator
technique is complicated by the difficulty of obtaining representative samples of
the food (i.e. pasture herbage) consumed. Animals graze selectively, preferring
young plants to old, and preferring leaf to stem, and a sample of the sward picked
by hand or cut with a mower is therefore unlikely to be representative of that
consumed by the animal. One way of obtaining representative samples is to use an
animal with an esophageal fistula (an opening from the lumen of the esophagus to
the skin surface).When this is closed by a plug, food passes normally between
mouth and stomach; when the plug is temporarily removed, herbage consumed
can be collected in a bag hung below the fistula. Samples of grazed herbage
obtained in this way can then be analyzed, together with samples of feces, for the
indicator. The n-alkane technique may also be useful in this context as there are
large and characteristic differences in the n-alkane content of different plant
species. By relating the pattern of faecal n-alkane output to the n-alkane pattern in
different plant species or parts, the technique allows estimates of the diet
composition of grazing animals to be made. 27
• C. Difference method
• This method is used when studying the digestibility of combination of feeds.
• Basal diet: When studying digestibility by difference, basal diet is the diet we want
to combine with our test feed. The objective is to determine the digestibility of the
test feed not the basal diet. To determine the digestibility of the test feed, first we
determine the digestibility of the basal diet, then combine the basal diet and test
feed and run digestibility trial and get digestibility of the test feed by difference. The
ration digestibility is the sum of digestive amounts of its components.
•
• Nutrient digestibility of test feed = A – (B – C)
• A
• Where:
• A = Nutrient in test feed
• B = Nutrient in the feces of mixed diets
• C = Nutrient in the feces from basal feed 28
Example: Estimate the digestibility of a 300 gram fish meal fed with 1 kg of basal diet barley?
  Phase II Digestibility trial for basal diet (barley) + test feed (fish meal)
Phase I Digestibility trial for basal diet (barley)

  Organic matter Protein   Organic matter Protein

           
In feed 850 gm 100 gm In feed 1060 gm 280 gm

In feces 150 gm 25 gm In feces 170 gm 35 gm

Digested 700 gm 75 gm Digested 890 gm 245 gm

%Digestibility 82% 75% Part Digested from barley 700 gm 75 gm

      Part digested from fish meal 190 gm 170 gm

      %Digestibility of fish meal 90% (A) 94% (B)

A = 190/(1060 – 850) x 100

= 90%
B = 170/(280 – 100) x 100
29
= 94%
• When digestibility is determined by the difference method one major problem is
the associative effect of feeds when they are combined. If the test feed and the
basal ration have a negative association, the overall digestibility may decrease. On
the other hand if the test feed has a positive correlation a better digestibility will
be obtained. It is hard to get an added effect of digestibility of nutrients when
more than one diet is combined. Therefore, errors will be there when one try to
estimate digestibility by difference.

30