Digestibility trial

Digestion and metabolism
trial
Dr. Ajith K.S. Ph.D.
Assistant Professor
Department of Animal Nutrition
College of Veterinary and Animal Sciences, Mannuthy

Digestion trial
 The amount of feed or nutrient intake by an animal which is
not excreted in the faeces is considered to be digested and
absorbed.
 For the determination of digestion coefficient/ digestibility, the
daily intake of feed/ nutrients and daily faecal void values
are to be recorded.

Metabolism trial
 the availability and retention of nitrogenous nutrients and
minerals is studied by conducting metabolism trial/ balance
or retention studies.
 The daily intake of feed/ nutrients is recorded.
 The routes of elimination considered during such metabolism
trial are faeces, urine, expired air, skin secretions, milk
(for lactating animals) and eggs (laying birds).
 In case of certain specific studies, materials like shed hair
and scurf are also collected.

Digestion trial vs. Metabolism trial
Particulars Digestion trial Metabolism trial
Purpose Gives information on proportion of nutrients
in a feed or diet that are absorbed from the
gastro intestinal tract
Similar to digestion trials but gives more
information on utilization of nutrient after
absorption from the gastro intestinal tract
Information obtained Information on digestibility co-efficient of
nutrients
In addition to digestibility co-efficient, one gets
information on nutrient balances such as
nitrogen, calcium, Phosphorus, energy etc.
Hence, metabolism trials provide complete
information on nutrient digestion and
utilization from feedstuffs than digestion trials
What is collected Only feces In addition to feces, urine, milk, gases like
co, sloughed hair, feathers etc are also
collected
Result Apparent digestibility co-efficient of
nutrients
In addition to apparent digestibility co-efficient
of nutrients, the information on positive or
negative nutrient balance is obtained

Methods of Determining Digestibility
 I. In vivo method
 The two in vivo methods are :
Direct Method or Conventional method
Indirect: In the indirect method there are two methods:
Difference Method
Indicators/Markers Method

II. Semi in vivo methods
 Nylon bag technique
 VIVAR technique ( In vivo artificial rumen technique)
 III. In vitro methods
 Using rumen liquor
 Using enzymes instead of rumen liquor
 RUSITEC method

 Measurement of digestibility by conventional method
 Digestibility of feeds/ nutrients is determined in vivo by
conducting digestion trials
 Procedure for conducting digestion/ metabolism trial
 Male animals are preferably used for conducting the trials
because it is easier to collect faeces and urine separately with
the male.

 Stages
 Pre-collection feeding period: in order to remove the effect
of previous feeding and also to adapt the animal on the
feed to be evaluated, the animals are fed the test feed for a
period of 2-3 weeks.
 Collection period: it may be 6 to 10 days for herbivores
like ruminants and 4 days for simple stomached animals
like pigs.

Methods followed and devices used
 Trial in stall: i facilities for offering feed and water to individual
animal
 In such stalls there will be provision for collecting the residues of
feed offered, faeces and urine.
 Trial in cages These metallic cages provide the animal adequate
space for standing and lying down but not turning back.
 A detachable feed box or trough is provided in front part of the
cage.
 A window is provided in the anterior half of the cage for offering
drinking water.

 The floor of the cage has a gradual slope towards the middle
to facilitate quantitative collection of urine
 Feaces will be collected manually, immediately after it is
voided by the animal.
 Digestion trial using faeces collection bag: a canvass bag
with rubber lining fitted on the animal is used for quantitative
collection of faeces.

 Digestibility
 Digestibility is defined as a portion of feed or any nutrient in feed which is absorbed
and not recovered in faeces.

 For example, if a cow ate 10 kg of hay containing 9 kg of dry matter and excreted 4
kg of dry matter in its faeces, the digestibility of the hay dry matter would be:

 The digestibility coefficient determined is apparent, since the faeces/dung contain
metabolic (mucosal debris, unspent enzymes, undigested microorganisms) as
well as undigested feed.
 Dung (digested DM excreted) = 3.7 kg from feed + 0.3 kg from body.
 Thus the apparent digestibility of feed is less than the true digestibility.

 The losses of the ingested carbohydrates as methane and
carbon dioxide are also accounted in digestibility. So
digestibility of carbohydrates is overestimated.
 Digestibility coefficients are estimated for all organic
nutrients.
 ash or minerals - not estimated, because it does not
contribute to energy to the feed, and most of the absorbed
minerals are excreted through the gut.

NORMS ADOPTED IN CONDUCTING
DIGESTION AND METABOLISM TRIALS
 Selection of animals
 The animals should be of the same breed, sex, age and
body weight.
 Generally a minimum of four adult animals are needed.
 Animals should be healthy and free from parasites.
 Male animals are preferred to females because it is easier to
collect faeces and urine separately.

Preliminary Period
 The test feed has to be fed daily in constant amounts, as per the requirements
of the animal, for an extended period. This is called preliminary period.
 The purpose is to make the gastrointestinal tract free of any indigestible
material from the feed consumed prior to the start of the digestion trial.
 In monogastric animals such as pigs, digestion and evacuation are usually
complete in 24 hours after the test feed is ingested.
 In ruminants eating a roughage ration, the last residues may not be voided until
150 to 200 hours (6 to 8 days) have elapsed, though 95% are usually voided
within 140 hours.
 So preliminary period of 2 - 3 days (some times 3 - 5 days) in pigs and 7 - 14
days (8 - 10 days) in ruminants is followed to eliminate the feed residues of
previous rations from the digestive tract and to stabilize the daily feed intake
and faecal output to a constant level.
 Water is provided at all times.
 Animals are fed individually

Collection period
 Animals are transferred to cages or stalls and a 2 - 3 day
adaptation period is allowed for their acclimatisation.
 A 5-7 day collection period is mostly followed.
 In general, the longer the period of collection the more
accurate the results
 Quantitative collection of faeces and urine are done.
 Representative samples of feed offered, feed leftovers and
faeces and urine voided are preserved and analysed for
nutrient composition.

Test feed
 The test feed should not be deficient in the nutrients because a deficiency of some
of them may affect digestion process.
 The test feed should be fed at the level required for meeting the requirements of the
animals.
 Normally 90% of the actual feed intake, as measured during the previous week, is
offered.
 Direct determination of digestibility of individual feed may be done if it provides a
satisfactory ration for the test period when fed alone.
 Some feeds cannot be fed alone as they do not supply the bulk and therefore their
digestibility coefficients cannot be determined directly. eg. Oilcakes, cereal grains,
concentrate mixtures.
 Similarly, non -maintenance type of roughages like straw, stovers, dried grasses,
etc. do not supply the required amounts of nutrients and so they cannot be fed alone to
the experimental animals.
 In the above mentioned cases digestibility is measured by indirect methods.

INDIRECT METHOD OF DETERMINING
DIGESTIBILITY : DIGESTIBILITY BY
DIFFERENCE
 Difference method
 When the digestibility of poor quality non-maintenance type of forage like stovers,
kadbi, straws or mature grasses is to be determined because these cannot be fed
as sole source of nutrients.
 If the digestibility of oilseed cakes, cereal grain and concentrate mixture is to
be determined then difference method is followed since these cannot be fed as
sole feed to ruminants.
 Procedure
 For example, if the digestibility of nutrients in a concentrate like maize grain or
groundnut cake and in a poor quality roughage like straw is to be determined then
three digestibility trials in a sequence are conducted.

 1st digestion trial
 feed the animals with a good quality roughage like legume hay e.g.
cowpea hay to determine the digestibility of nutrients in it.
 2nd digestion trial
 the same animals are fed the same good quality fodder i.e. cowpea hay that
was fed during first digestion trial along with known quantity of concentrate
like groundnut cake or maize grain etc whose digestibility is to be estimated by
difference.
 3rd digestion trial
 by feeding the same animals with the concentrate whose digestibility was
estimated in the second trial along with the poor quality roughage i.e. grass
whose digestibility is to be determined. The digestibility of poor quality
roughage will be determined by difference using predicted value of groundnut
cake with the help of earlier trials (a) and (b).

INDICATOR METHOD OF DETERMINING
DIGESTIBILITY
 In some circumstances the lack of suitable equipment of the particular nature of
the trial may make it impracticable to measure directly either food intake or faeces
out put, or both. For instance, when animals are fed as a group it is impossible to
measure the intake of each individual.
 Digestibility can still be measured, however, if the food contains some substance
which is known to be completely indigestible.
 If the concentrations of this indicator substance in the food and in small
samples of the faeces of each animal are then determined, the ratio between
these concentrations gives an estimate of digestibility.
 For example, if the concentrations of the indicator increased from 1% dry matter
to 2% in the faeces, this would mean that 50% of the dry matter had been
digested and absorbed.

 The indicator may be a natural constituent of the food or be a chemical mixed
into it. It is difficult to mix chemicals with foods like hay, but an indigestible
constitutent such as lignin may be used.
 Other indicators in use today are fractions of the food known as indigestible acid-
detergent fibre and acid insoluble ash (mainly silica) and also some naturally
occuring n-alkanes of long chain length (C25- C35).
 The indicator most commonly added to foods is chromium in the form of chromic
oxide, Cr2O3.Chromic oxide is very insoluble and hence indigestible; moreover,
chromium is unlikely to be present as a major natural constituent of foods.

 For non ruminants ,titanium dioxide may be added to foods as an indicator.
 Chromic oxide may be used as an indicator in a different way, to estimate faeces
output rather than digestibility.
 In this application the marker is given for 10 -15 days in fixed amounts ( eg.
administered in a gelatin capsule) and once its excretion is assumed to have
stabilised its concentration in faeces samples is determined. Faeces dry matter
output (kg/day) is calculated

 Marker dose (g per day)/ Marker concentration in faeces
DM (g/kg).
 For example, if an animal was given 10 g of chromic oxide
per day and the concentration of the marker was found to be
4 g/kg faecesDM, faeces output would be calculated as 10/4
=2.5 kg DM/day.
 If food intake was known, dry matter digestibility could be
calculated

The ideal specification of an indicator/marker
 It should be totally indigestible.
 It should not have any pharmacological action on the digestive tract. It should
be inert to the digestive system.
 It must mix intimately and remain uniformly distributed in the digesta.
 It should pass through the tract at a uniform rate and should be voided entirely.
 It can readily be determined chemically, and
 Preferably be a natural constituent of the feed under test.

Indicators may be used to measure digestibility of
feed under the following circumstances
 If metabolism cages and other facilities for direct
collection of feces and urine voided are not available
 If animals are fed in groups, then it is impossible to record
the feed consumed and feces voided by each animal in the
group.
 To know intake of herbage from cultivated or natural
pastures and digestibility of nutrients in the pasture
consumed by the animal.

MEASUREMENT OF PASTURE
CONSUMPTION AND DIGESTIBILITY IN
GRAZING ANIMALS
 It is difficult to obtain, the representative sample of forage actually eaten by the
grazing animal and quantitative collection of faeces by faeces bag.
 Therefore markers have been used for both the determination of digestibility of
pasture herbage and DMI through grazing.

 Digestibility can be determined through use of an internal indicator.
 Faecal output is measured concurrently by using an external marker and intake is
calculated as follows.
 Normally chromic oxide is fed in a capsule to the grazing animals and the number of
grab samples of faeces are taken at different time interval to determine the average
concentration of the indicator per unit weight of faeces.


Use of markers
 Measurement of digestibility coefficients without total faecal collection
 Measurement of herbage intake in grazing animals
 Markers are used for quantifying the rate of passage and extent of digestion in
different segment of the gut.
 Rare earths (Lanthanam,Samarium,cerium,ytterbium and dysprosium) may be
used as reliable markers of particulate phase of digesta.
 Polyethylene glycol (PEG), Chromium, EDTA and Cobalt EDTA are liquid
phase markers in ruminant studies.

LABORATORY METHODS OF
ESTIMATING DIGESTIBILITY
 Since digestibility trials are laborious to perform, there have been numerous
attempts made to determine the digestibility of foods by reproducing in the
laboratory the reactions which take place in the alimentary tract of the animal.
 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.

Tilley and Terry method
 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 a known weight of the finely ground sample of the feed whose
organic matter composition is already determined is incubated for 48 hours with
buffered rumen liquor in a tube under anaerobic conditions.
 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.
 The insoluble residue is filtered off, dried and ignited and again weighed.
 The difference between the two weighing gives the organic matter present in the
residue.
 The digestibility coefficient determined in vitro is generally 1-2 percentage units lower
than the coefficient measured in vivo.

In sacco or In situ or Semi in vivo or
Nylon or Dacron Bag Technique
 The digestibility/degradability of feeds in the rumen can be determined by keeping
the feed sample in bags which are immersed in rumen contents of rumen
fistulated animals.
 The bags are made up of nylon, dacron or silk cloth which is indigestible and
should be of very fine mesh so that the test feed particles should not pass out of
the bag undegraded but at the same time it should allow the rumen microbes to
enter into the bag and act on the test feed.
 The bags on removal at different time intervals are washed till the wash water is
clear and dried at 600C for 48 hours.
 The percent disappearence of dry matter, nitrogen/crude protein, different
fibre fractions etc are determined.

Applications of the technique
 This technique provides a powerful tool for initial evaluation of
feedstuffs and is useful in screening, rapidly, large number of
samples developed in forage breeding experiments .
 This technique is helpful to understand the rumen processes. It is
possible to vary the factors within the bag or within the rumen.
 Alternatively, the conditions within the rumen ie: rumen environment,
can be varied and a standard material incubated in the bag in order to
study the effect of rumen environment on the rate of degradation.

Limitations
 The test feed in the bag is not subjected to the total ruminal
experience, ie., mastication, rumination and passage.
 What is actually measured is the breakdown of material to a
size small enough to leave the bag and not necessarily a
complete degradation to simple chemical compounds.

In Vivo Artificial Rumen (VIVAR) Technique
 studying nutrient utilisation by rumen micro-organisms under controlled
conditions in the rumen.
 The system consists of a porcelain test tube or stainless steel or glass jars
fitted with bacteriological membranes to provide controlled interchange of the
rumen contents.
 The rumen microflora pass through the semipermeable membranes and
degrade feed samples present inside the VIVAR tube, but the sample
particles cannot move outside. After completion of the fermentation period,
VIVAR tubes is removed.
 The dry matter disappearance will be recorded by difference in weight of the
sample and the residues left in the VIVAR tube.

In vitro gas production
 In vitro gas production (Menke et al., 1979): gas production
rather than dry matter loss is measured.
 The amount of gas (carbondioxide and methane) released
when feeds are incubated at 390C inside a calibrated glass
syringe with rumen liquour is closely related to digestibility
of feed. The feeds of different digestibility produce different
volume of gases within a stipulated time.

The rumen simulation technique
(RUSITEC)
RUSITEC is an apparatus developed for artificially
creating conditions somewhat similar to those in
the rumen.
With the RUSITEC it is possible to determine all the
inputs and outputs’ including gases to investigate
what goes on inside the reaction vessel.

FACTORS AFFECTING DIGESTIBILITY
OF FEED
A. ANIMAL FACTORS
B. CHEMICAL COMPOSITION OF THE FEED
C. PREPARATION OF FEED

ANIMAL FACTORS
 Species
 Roughages high in crude fibre are better digested by ruminants than by non-
ruminants due to the pre-gastric fermentative digestion that occurs in the
ruminants.
 In several non-ruminants, post-gastric fermentative digestion occurs which
helps in digestion of crude fibre.
 Pre-gastric fermentative digestion is highly efficient since the nutrients
released are digested and absorbed in stomach and small intestine.
 The ruminant is more efficient in the utilization of high-fibre, low protein
forage; whereas the simple stomached pig is more efficient in utilization of high
protein, low-fibre feedstuffs.

Age
 Very young or very old animals are usually less efficient in their digestion of
feeds.
 The young ruminants can neither eat nor digest roughage until their digestive
tracts, specially their rumens are developed.
 Digestibility of fat in chickens is higher in adults than in young chicks.
 In case of old animals their ability to chew feed is impaired by worn out teeth.
 Declining health and reduced secretion of enzymes may adversely affect the
digestibilty at an advanced age.

Work
 Light work seems to improve digestibility of feeds, while heavy work
depresses it.
Individuality
 Individual variation of as much as 25% has been observed in the digestive ability
of the same feed among animals. However, most animals have shown variation of
about 4 - 5%.
Level of feeding
 Generally when more feed is consumed by the animal the rate of passage of
the digesta in the alimentary canal is faster and the digestibilty declines due
to lesser retention time.

CHEMICAL COMPOSTION OF FEED
 Generally grains are well utilised by all classes of livestock.
 The digestibility of forages is closely related to the chemical composition.
 The chemical composition of the forage is affected by number of factors like soil
composition, manuring and fertilization, water supply, stage of maturity of
the plant, frequency of cutting, variety and strain of the plant, climate, etc;
the predominant factor being the stage of maturity when cut.
 Differences among varieties within the same species may be due to the physical
composition of the plant.ie: leaf to stem ratio, soil fertility, etc. Early cut fodder
has higher digestibility than late-cut.
 The protein, minerals and vitamins decreases while crude fibre increases as
the plant matures.

PREPARATION OF FEED
 Particle size of the feed
 Grinding of grains and other feed helps to improve digestibility in young
piglets with undeveloped teeth and in older animals with worn out teeth.
 In general grinding increase digestibility, because of increase surface area for
enzymatic action and disruption of grain coat. If grain or any other feed is ground
to a fine particle size, the feed is less palatable and digestible
 If roughages are ground to fine grinding, digestibility of fibre is decreased
while total consumption is increased due to increased rate of passage.
 Rumen fermentation pattern is also changed due to fine grinding of feed.

Soaking
 Soaking of grains and feed in water before feeding generally
increases digestibility.
 Processing of grains/feed
 Processing by boiling, steam processing, micronization,
pelleting, extrusion cooking, improves their digestibility.
 Some processing methods depress digestibility due to
increased dry matter consumption and faster rate of
passage. This is more conspicuous in pelleting of
roughages, where digestibilty of DM and crude fibre
decreases.

Nutrient content in the ration/ration
composition
 Protein level: This "associative effect" of feeds on one
another's digestibility is more evident in the case of ruminants,
when the addition of a protein or NPN compound to a low
protein ration increases the microbial digestion of the
crude fibre by stimulating the growth of microorganisms
in the rumen.
 As the dietary protein level increases, the digestibility of all
the nutrient increases. Similarly, as the dietary protein level
is lowered ,the digestibility of all the nutrients decreases.

 Carbohydrates: The nature and level of dietary carbohydrates
affect the digestibility of all nutrients present in the diet.
 In ruminants, excessive levels of soluble carbohydrates
(eg.molasses 7% and above) results in lower microbial
breakdown of crude fibre.
 It tends to depress not only the digestibility of cellulose,
hemicellulose, etc., but other nutrients also.
 The higher the percentage of crude fibre in a ration, the lower
is the digestibility of dry matter and all other nutrients.

 Lipids: Addition of oil or fat in a diet increases the
digestibility coefficient of ether extract, as such fats have
higher digestibility than other constituents of the ether
extract.
 Higher levels of fat in the diets generally reduce the
digestibility of other nutrients, particularly of dietary fibre.

 Minerals: In the diets of pigs and poultry, mineral content
does not seem to influence the digestibility of other
dietary constituents.
 Deficiency of minerals in herbivorous animals limits the
growth of microorganisms and this will reduce the
digestibility of crude fibre and of other nutrients as well.
 Adequate amount of salt and water tend to improve
digestibility.

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