Digestibility Trials

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 valueDigestible 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

20 40 60 80 100

200

400

600

800

Test feed digy.

Base feed digy.

% inclusion of test feed in ration

DM

D (

g/k

g)

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 = 100 – 100 x % indicatorfeed X % nutrientfeces

Digestibility % indicatorfeces % 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

experimentsAnimal 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)

Method 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

(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)

r2 =0.24

530 580 630 680

Rumen fluid-pepsin DOMD

530

550

570

590

610

630

650

670

690

In v

ivo D

OM

D

(Adesogan et al. 1998)

Year One Year Two

Rumen fluid technique -

problemsStandards 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

Method 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

(Givens et al., 1990)

Poorer relationships found for autumn grass (r2 = 13- 20)

Prediction of in vivo OMD of spring

grass from different methods

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 hydrolysis (mg/ml)

0.0 10 20Time (h)

0.00.25

0.5 Co-culture

S. bovis

S. ruminantium

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 true DOMD from bag true DOMD

y = 0.99x + 3.61

r2

= 0.93; rsd=2.93

50

60

70

80

50 55 60 65 70 75 80 85bag

tub

e

Prediction of tube app. DOMD from bag app. DOMD

y = 0.87x + 4.25

r2

= 0.83; rsd = 4.04

40

50

60

70

80

40 50 60 70 80bag

tub

e

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

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. 113-

134.

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.

Digestibility references