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DIGESTIBILITY

Digestibility Trials

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Page 1: Digestibility Trials

DIGESTIBILITY

Page 2: Digestibility Trials

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?

Page 3: Digestibility Trials

In vivo digestibility methods

Direct or total/complete collection

Difference method

Regression method

Indirect method

Page 4: Digestibility Trials

1. Total collection

Page 5: Digestibility Trials

In vivo digestibility trials in

metabolism crates

Page 6: Digestibility Trials

In vivo digestibility trials in pens

Page 7: Digestibility Trials

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

Page 8: Digestibility Trials

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)

Page 9: Digestibility Trials

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

Page 10: Digestibility Trials

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.

Page 11: Digestibility Trials

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)

Page 12: Digestibility Trials

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

Page 13: Digestibility Trials

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

Page 14: Digestibility Trials

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.

Page 15: Digestibility Trials

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

Page 16: Digestibility Trials

Marker types

External

– Chromic oxide

– Dysporium

– Polyamide

Can contaminate

forage

Internal

– Lignin

– AIA

– ADF

– n-alkanes

Easier, less labor

Page 17: Digestibility Trials

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

Page 18: Digestibility Trials

Problems with in vivo

experimentsAnimal trials are:

– Expensive

– Protracted

– Laborious

– Public concerns

– Animal stress ???

Must estimate nutritive value with less animal

dependent techniques

Page 19: Digestibility Trials

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

Page 20: Digestibility Trials

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

Page 21: Digestibility Trials

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)

Page 22: Digestibility Trials

Rumen fluid problems

Variation in Inoculum composition & activity due to

– Host animal diet

– Animal species

– Collection time

– Processing (blending vs. filtration)

Page 23: Digestibility Trials

Rumen fluid problems

Analytical issues

– Maintenance of anaerobic media; optimal pH, temp

– High viscosity hinders filtration

– Offensive odors

– Hygiene – (Prevent pathogen infection)

Page 24: Digestibility Trials

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

Page 25: Digestibility Trials

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

Page 26: Digestibility Trials

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

Page 27: Digestibility Trials

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)

Page 28: Digestibility Trials

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

Page 29: Digestibility Trials

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)

Page 30: Digestibility Trials

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

Page 31: Digestibility Trials

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

Page 32: Digestibility Trials

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)

Page 33: Digestibility Trials

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

Page 34: Digestibility Trials

Enzyme method problems

Equations are species-specific

Represent effect of a few enzymes

Variability in enzyme activity

– Due to enzyme source & batch

Page 35: Digestibility Trials

The ANKOM equipment

Page 36: Digestibility Trials

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

Page 37: Digestibility Trials

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

Page 38: Digestibility Trials

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

Page 39: Digestibility Trials

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