DIGESTIBILITY
Apparent v. true digestibility
True digestibility involves correction for endogenous losses,
apparent digestion does not.
Endogenous losses
– Include:
• Sloughed off intestinal cells
• Digestive juices (enzymes)
• Microbial matter
– Quantified by measuring fecal output of fasted animals
– Can be 9.8 to 12.9 % DMI
– Should they be quantified?
In vivo digestibility methods
Direct or total/complete collection
Difference method
Regression method
Indirect method
1. Total collection
In vivo digestibility trials in
metabolism crates
In vivo digestibility trials in pens
Total collection
calculations
Digestibility (g/kg) =
Nutrient in feed - Nutrient in feces x 1000
Nutrient in feed
Dry matter digestibility (DMD, g/kg) =
DM in feed - DM in feces x 1000
DM in feed
Organic matter digestibility (OMD, g/kg) =
OM in feed - OM in feces x 1000
OM in feed
Can be expressed as a proportion, % or g/kg
Digestibility indices that estimate
energy value
Digestible organic matter content (DOMD) (g/kg DM)
= OM in feed - OM in feces
x 1000
DM in feed
TDN = DCP + DCF + DNFE + DEE(2.25)
– DCP= Digestible Crude Protein
– DCF= Digestible Crude Fiber
– DNFE= Digestible Nitrogen-Free Extract
– DEE= Digestible Ether Extract (2.25)
2. Difference method
Allows digy calculation for 2 feeds fed simultaneously
Assumptions
– No interaction b/w the digy of the feeds
– Must know digy & fecal DM output (DMO) of base
feed
Test feed DMD =
Test feed DMI – (Fecal DMO- Base feed DMO)
Test feed DMI
Cons
– Assumptions may be invalid
3. Regression method
Schneider & Flatt (1975)
Also allows digy. estimation for two feeds
– Feed different ratios of the two feeds
– Estimate digy of each of the ratios
– Fit regression of test feed inclusion vs. digy
– Extrapolate to estimate digy of test feed.
Cons
– Considerable expense and labor for estimating digy
of one feed.
Regression method
DMD (g/kg)
800
Base feed digy.
600
400
Test feed digy.
200
20
40
60
80
% inclusion of test feed in ration
100
Digy trial issues
Changeover designs
– necessary if period effects are an issue e.g.
• Animal physiological changes
• Forage physiological changes
Adaptation period
– Necessary to adapt the animals to
• New feed (microbial population changes)
• Strange equipment
• Strange housing
– 6 – 14 day period is the norm
Marker digestibility trials
Particularly useful for grazing animals
Procedure
– Add indigestible marker to feed eg chromic oxide
– Measure concentration in feed & feces
– Estimate disappearance of marker from gut.
E.g. if a feed contains 1% Cr2O3 & feces contains 2%
Cr2O3, diet digestibility = 50%
– Since Cr3O2 conc. has doubled, 50% of DM must have
been digested
Marker trials contd.
For the digy of a specific nutrient,
must also know the % nutrient in feed & feces
%Nutrient
Digestibility
=
100 – 100 x % indicatorfeed
% indicatorfeces
X
% nutrientfeces
% nutrientfeed
Homework:
If lambs are fed a bahia grass diet containing 7%
protein & 1% chromic oxide, and their feces contains
5% CP and 2% chromic oxide. Calculate CP digy.
Marker digestibility
Pros
– Total feces collection not necessary
– Total intake determination not necessary
– Easier, less labor
Cons
– Representative sampling essential
– Accurate estimation of nutrient or marker conc.
essential
– Assumes complete excretion of marker hence
Recovery of marker determines accuracy of digy
Marker types
External
– Chromic oxide
– Dysporium
– Polyamide
Can contaminate
forage
Internal
– Lignin
– AIA
– ADF
– n-alkanes
Easier, less labor
Marker issues
Difficulty of mixing marker with forages
– Dose cows instead- ( s handling)
Marker migration
– Must not affect feed digy
External markers may contaminate forage
Problems with in vivo
experiments
Animal trials are:
– Expensive
– Protracted
– Laborious
– Public concerns
– Animal stress ???
Must estimate nutritive value with less animal
dependent techniques
Ideal in vitro methods should be:
– Rapid (one step) & routinely practicable
– Accurate
– Cheap & not laborious
– Repeatable & robust
– Biologically meaningful
– Broad-based (apply to all forage types)
– Handle large nos. of samples
– Laboratory-based
Rumen fluid –pepsin in vitro
digestibility (IVOMD)
•Developed by Tilley & Terry
(1967)
•Measures apparent digy in rumen
fluid (48 h) and acid pepsin (48 h)
•Gives accurate predictions of in
vivo digy for most forages
Prediction of silage OMD in vivo from
different methods (g/kg DM)
r2
RSD
KMnO4 lignin
21.8
54.6
ADF
32.1
50.9
NDF
45.7
45.5
(M) ADF
55.8
40.9
IVOMD
74.1
33.6
Method
(Givens et al., 1989)
Rumen fluid problems
Variation in Inoculum composition & activity due to
– Host animal diet
– Animal species
– Collection time
– Processing (blending vs. filtration)
Rumen fluid problems
Analytical issues
– Maintenance of anaerobic media; optimal pH, temp
– High viscosity hinders filtration
– Offensive odors
– Hygiene – (Prevent pathogen infection)
Relationship between in vivo and
in vitro DOMD of wheat silage (g/kg DM)
690
Year One
In vivo DOMD
670
Year Two
650
r2 =0.24
630
610
590
570
550
530
530
580
Rumen fluid-pepsin DOMD
630
680
(Adesogan et al. 1998)
Rumen fluid technique problems
Standards needed to correct for variability in rumen
fluid composition & activity
Disregards / inappropriately represents:
– Ruminal outflow (uses a batch process)
– Digests maillard product not digested in vivo
– Associative effects between feeds
– Endogenous secretions
– Post abomasal digestion
Alternatives to Tilley & Terry
1. Rumen fluid – Neutral detergent (Van Soest, 1967)
– More akin to true digestibility
– Gives higher digy. values
– Still requires rumen fluid
2. Feces
– Gives lower digestibility estimates
3. Enzyme- based assays
Prediction of DMD in vivo from in vitro
fecal liquor DMD
Spp. of feces donor
r2 range
Ovine
0.33 – 0.98
Bovine
0.77 – 0.97
Equine
0.90
Caprine
0.96-0.97
(Ohmed et al., 2001)
Cell-free enzyme in vitro digestibility
Examples of procedures used:
1. Cellulase
2. Neutral detergent- cellulase
3. Neutral detergent-cellulase +gammanase
4. Pepsin cellulase
Amylase pre-treatment important for starch-rich feeds
Gammanase for oil-rich feeds
Relationships between DMD in vivo and
enzyme predicted DMD
Method
R2
Cellulase
0.83
Neutral detergent cellulase
0.94
Acid pepsin – cellulase
0.88
Rumen fluid
0.83
(Bughara & Sleper, 1986)
Prediction of in vivo OMD of
forages from different methods
Method
r
RSD (%)
AE(+)
ND + cellulase
0.90
3.3
0.9
Pepsin + cellulase
0.94
2.6
0.3
(McLeod & Minson, 1982)
Higher analytical error with ND – cellulase technique
may outweigh shorter processing time
Prediction of in vivo OMD of spring
grass from different methods
r2
RSD
ND + cellulase
76.6
27.1
Pepsin + cellulase
75.9
28.8
Rumen fluid-pepsin
67.0
33.2
(M) ADF
66.9
33.3
Method
Poorer relationships found for autumn grass (r2 = 13- 20)
(Givens et al., 1990)
Effect of enzyme source on cellulase
activity
% DM solubilized
Fungi
Herbage
Cellulose paper
Trichoderma spp.
57
69
Basidiomycete
48
20
Aspergillus niger
45
10
Rhizopus spp.
35
7
(Jones & Hayward, 1975)
14C-Casein
0.5
hydrolysis (mg/ml)
Co-culture
0.25
0.0
S. bovis
S. ruminantium
0.0
10
Time (h)
20
Commercial enzymes don’t fully simulate microbial
activity of mixed rumen microbes
Enzyme method problems
Equations are species-specific
Represent effect of a few enzymes
Variability in enzyme activity
– Due to enzyme source & batch
The ANKOM equipment
Ankom digestibility validation
Prediction of tube app. DOMD from bag app. DOMD
Prediction of tube true DOMD from bag true DOMD
80
y = 0.87x + 4.25
80
2
r = 0.83; rsd = 4.04
y = 0.99x + 3.61
2
70
r = 0.93; rsd=2.93
tube
tube
70
60
60
50
50
40
40
50
bag
60
50
70
80
55
60
bag
65
70
75
80
85
ANKOM pros & cons
Pros
– Simplifies filtration, incubation and mixing
– Uses a batch process (& ash-free bags)
Cons
– Bag pore size may allow excess outflow or restrict
microbial colonization
– Bag material & pore size may affect results
• Monofilamentous cloth – precise aperture
• Multifilamentous cloth – pore size affected by stresses
e.g. dacron
In vitro digestibility summary
Pros
– Predicts in vivo digy more accurately than NDF or
lignin
– Handles several samples & are biologically
meaningful
Cons
– May require fistulated animals
– Labor intensive & protracted
– Plagued by variability in composition & activity of
inoculum/enzyme
– Doesn’t indicate the kinetics of digestion
Digestibility references
Chapters 6 – 8 In: D.I. Givens, E. Owen, R.F.E. Axford and H.M. Omed (Editors) 2000,
Forage Evaluation in Ruminant Nutrition. CABI Publishing, Wallingford, UK, pp. 113134.
Adesogan, A.T, Givens D.I. and Owen. E. Measuring chemical composition and nutritive
value in forages. Field and Laboratory methods for grassland and animal production
research. CABI Publishing. P 263
Tilley, J.M.A. and Terry, R.A., 1963. A two stage technique for the in vitro digestion of
forage crops. Journal of the British Grassland Society, 18: 104-111.
Van Soest, P.J., Wine, R.H. and Moore, L.A., 1966. Estimation of the true digestibility of
forages by the in vitro digestion of cell walls. Proceedings of , The Xth International
Grassland Congress, Helsinki. Finish Grassland Association., pp 438-441.
Vogel, K.P., Pedersen, J.F., Masterson, S.D. and Toy, J.J., 1999. Evaluation of a filter bag
system for NDF, ADF, and IVDMD forage analysis. Crop Science, 39: 276-279.
Wilman, D. and Adesogan, A., 2000. A comparison of filter bag methods with conventional
tube methods of determining the in vitro digestibility of forages. Animal Feed Science and
Technology, 84: 33-47.