In Situ Rumen

Degradability Methods

,

Department of Animal Sciences,

University of Florida

Dr Gbola Adesogan

In situ rumen degradability (ISD)

Determines the disappearance of feeds incubated in

a porous bag within the rumen

Measures degradability (≠ digestibility)

(digested feeds > pore size not considered degraded)

Estimates the extent & rate of degradation

Basis of formulating rations to meet protein

requirements of livestock in many countries

In situ degradability graph

b

Time (h)

a

24 7212 36 480

c

Degradability (g/kg)

In situ degradability calculations

a= zero time intercept

b = slowly degradable fraction

Degradability = a + b ( 1 – ect)

c = degradation rate

t = time

To account for feed outflow from rumen, (which reduces

the actual rumen degradability ) we use

‘Effective’ degradability = p =a+(b x c)/ (c+kp)

Where kp= rate of passage (%/h)

When a, b & c values are generated

for protein dissappearance:RDP = a + b (c/c+kp

)

UDP = b [kp/ (c + kp)]

Calculate the RDP and UDP for these feeds

a, % b, % c, /h

Fishmeal 13 77 0.01

Ryegrass silage 63 26 0.14

Soybean meal 8 90 0.11

Accuracy (r2) of predicting

digestibility & intake from ISD

Factors Digestibility DM intake

a + b 0.82 0.77

(a+b) + c 0.86 0.88

(Khazaal et al., 1993)

0.8Grass silage

Time (h)

N d

egra

dab

ilit

y

0.6

0.4

0.2

048

hay

2412 36

Differences in the N

degradability & a and

b fractions of feeds

0.8 Soyabean meal

Time (h)

N d

egra

dab

ilit

y

0.6

0.4

0.2

048

fishmeal

2412 36

Urea

Benefits of knowing feed N

degradability

Allows partitioning of protein sources according to

whether they contain predomininantly:

1. Rumen degradable protein (RDP)E.g. soybean meal, alfalfa,

2. Undegradable protein (RUP or UDP)E.g Fish meal, bone meal,

Benefits of knowing the N

degradation of feeds

Allows UDP supplementation at high performance levels

Allows formulation of diets to ensure synchronous

ruminal supply of energy and protein

1. Feed readily degradable N feeds with readily

fermentable energy sources e.g.

• Soybean meal and grass silage

2. Feed undegradable N feeds with feeds high in slowly

fermentable energy e.g

• Hay / maize silage and fishmeal

Factors affecting degradability results

Host animal spp & diet.

Sample processing

Particle size / form / fine particle losses

Sample size to surface bag area ratio

Bag pore size

Data modelling

Microbial N contamination of bags

Incubation sequence

Effect of host animal on wheat silage

degradation

Host animal should be identical to those that will receive the test feed

40

45

50

55

60

65

70

75

80

0 20 40 60 80 100

DM

dis

appea

rance

(%)

s

s

ss

s

s s

c

cc

c

c

cc

Time (h)

Effect of animal spp. on rumen degradability

in WCW

DM

dis

ap

pea

ran

ce (

%)

Time (h)

40

45

50

55

60

65

70

75

80

0 20 40 60 80 100

s

s

ss

s

s s

c

cc

c

c

cc

c= cow

s=sheep

(Adesogan et al., 1998)

Host animal diet

Determines ruminal microbial composition

Recommendations

– Ensure diet is balanced

– Feed it at level that supports target production level

– Ensure diet & test feed are ……………………….

Sample size to bag surface ratio

Overfilling bags

– Delays bacterial attachment & reduces. digestion

Underfilling bags

– May leave insufficient residue for analysis

Ideal ratio

= 10 – 15 mg/cm2 of bag surface area

Note : Count both bag sides (leave room for seal)

e.g 4 g of DM weighed into an 8 x 14 cm bag = 4000 mg/224cm2 = 17.9 mg/cm2

Pore size

Varied sizes in literature (< 15 µm to 52 µm) due to:

Conflicting aims:

– Maximising bacterial colonization & fluid ingress

– Minimizing loss of undigested substrates

Implications

– Variable particulate losses

– Pressure build up which decreases

digestibility.

Effect of washing procedure

Forage type Parameter

Corn silage a 0.48 0.16

“ p 0.79 0.47

Grass silage a 0.31 0.16

Washing procedure

Machine Manual

“ p 0.62 0.47

Implication: concentrates, starch-rich feeds are susceptible to

fine particle losses which overestimate degradability

Microbial N contamination of bags

Underestimates degradability in low N, high fiber

feeds (can be up to 25%

Less important in concentrates (< 10%) high in CP

Causes erroneous lag and rate estimates

Solution : remove microbes by

• Thorough washing / Dip in ice

• DAPA / nucleic acids

• Correction equations

• Sonication / Stomaching

Cloth type & weave pattern

Monofilamentous vs. multifilamentous

In monofilamentous mesh types, pore sizes are more

prone be rearranged by stresses

Polyester fabric is preferable to dacron

In situ method - SummaryBiologically it is the most meaningful & accurate method for estimating kinetics of digestion

However

Difficult to standardize & laborious

Has low reproducibility

Inaccurate for soluble or small particulate feeds

Requires fistulated animals

Handles few samples & protracted

Loss of soluble non-degradable matter

Innacurate for estimating the effect of anti-nutrients

Recommend in situ procedures

Diet 60:40 hay:concentrate

Feeding level Maintenance

Bag material & pore size Polyester, 40 -60 μm

Sample size: bag area 10-15 mg/cm2

Particle size > 2 mm

No. of replicate animals > 2

Incubation sequence All in, sequential removal

(with machine washing)

Microbial correction Yes, if detectable

(Broderick & Cochran, 2000)

Other degradability methods

contd.Incubation in rumen fluid

– Labour intensive

– Involves animal experimentation except if rumen fluid

sourced from abattoirs

– History of abattoir rumen fluid unknown

– Batch culture

Incubation in

– Buffers

– Proteolytic enzymes

Buffer degradability methods

MacDougal’s buffer solubility

Burrough’s buffer solubility

Saline solutions

TCA precipitation

Tungstic acid precipitation

Cold water solubility

0.54Autoclaved rumen fluid

0.470.15M NaCl

0.66Burroughs buffer

rMethod

Correlation between selected buffer methods and in situ degradability

Buffer methods - summary

Pros

– Simple to use

– OK for ranking e.g. effect of heat treatment

– OK for estimating ‘a’ fraction

– OK if good relationship b/w solubility & degradability

• albumin is soluble but not easily degradable

• Casein is degradable but not readily soluble

Buffer methods - summary

Cons

– Imprecise estimates of degradability especially for forages

Equations are species-specific,

Precipitation methods measure true protein, yet ruminants also use NPN

Do not accurately estimate degradation rates

Enzyme-based degradabilityBacterial

Bacteroides amyliphilus

Streptomyces griseus

Bacillus subtilis

Plant proteases

Papain & bromelain

Fungal proteases

Aspergillus oryzae

Animal proteases

Pancreatin & pepsin

In-situ versus protease degradability

R2 Reference

S. Griseus (n= 21) 0.79 Aufrere & Cartailler ‘88

Ficin (n=38) 0.85 Kosmala et al. (1996)

Bromelain (n=41) 0.53 Tomankeva et al. 1995

Bromelain (n=68) 0.55 Tomankeva et al. 1995

(Broderick, 1999)

Little success in predicting the rate of degradation

14C-Casein hydrolysis (mg/ml)

0.0 10 20Time (h)

0.00.25

0.5 Co-culture

S. bovis

S. ruminantium

Single proteases can’t fully simulate microbial

activity of mixed rumen microbes

Degradability referencesBroderick and Cochran 2000. In vitro and in situ methods for measuring

digestibility with reference to protein degradability. In: Feeding Systems and feed evaluation models. M.K. Theodorou and J. France. (Editors). CABI Publishing.

Noziere, P. and Michalet-Doreau, B., 2000. In sacco methods. In: J.P.F.D'Mello (Editor), Farm animal metabolism and nutrition. CABInternational, Wallingford, pp. 233-254.

Huntington, J.A. and Givens, D.I., 1995. The in situ technique for studying therumen degradation of feeds: A review of the procedure. Nutrition Abstractsand Reviews (Series B), 65: 65-93.

Orskov, E.R., 2000. The in situ technique for the estimation of foragedegradability. In: D.I. Givens, E. Owen, R.F.E. Axford and H.M. Omed(Editors), Forage Evaluation in Ruminant Nutrition. CABI Publishing,Wallingford, UK, pp. 175-188.

Orskov, E.R., Hovell, F.D.D. and Mould, F., 1980. The use of the nylon bagtechnique for the evaluation of feedstuffs. Tropical Animal Production, 5:195-213.

Adesogan, A. T. Givens, D. I, and Owen, E. 2000. Chemical composition andNutritive Value of Forages. Field and Laboratory Methods for Grasslandand Animal Production Research (eds L t’Mannetje and R M Jones) pp 263-278. CABI Publishing. Wallingford UK