In Situ Rumen Degradability Methods - Department of Animal ...

In Situ Rumen
Degradability Methods
Dr Gbola Adesogan
,
Department of Animal Sciences,
University of Florida

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
Degradability (g/kg)
c
b
a
0
12 24 36 48 72
Time (h)

In situ degradability calculations
Degradability = a + b ( 1 – e ct )
a= zero time intercept
b = slowly degradable fraction
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+k p )
Where k p = rate of passage (%/h)

When a, b & c values are generated
for protein dissappearance:
RDP = a + b (c/c+k
)
p
UDP = b [k p / (c + kp)]
a, % b, % c, /h
Fishmeal 13 77 0.01
Ryegrass silage 63 26 0.14
Soybean meal 8 90 0.11
Calculate the RDP and UDP for these feeds

Accuracy (r 2 ) 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)

N degradability
N degradability
0.8
0.6
Grass silage
hay
Differences in the N
degradability & a and
b fractions of feeds
0.4
0.2
Urea
0
12 24 36
Time (h)
48
0.8
Soyabean meal
0.6
fishmeal
0.4
0.2
0
12 24 36
Time (h)
48

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

DM disappearance (%)
DM disappearance
(%)
Effect of host animal on wheat silage
80
75
70
65
60
55
50
45
40
s
c
cs
degradation
Effect of animal spp. on rumen degradability
80
in WCW
c
c
75
c
c
s
s
s
70 c s s s
c
65 c
c c= cow
60 s
cs
s
55
c
s=sheep
s
50 cs
s
c
45
40
0 20 0 4020 4060 60 80 80 100 100
(Adesogan et al., 1998)
Time (h)
Time (h)
Host animal should be identical to those that will
receive the test feed

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/cm 2 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/224cm 2 = 17.9 mg/cm 2

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
Washing procedure
Machine
Manual
Corn silage a 0.48 0.16
“ p 0.79 0.47
Grass silage a 0.31 0.16
“ 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 - Summary
Biologically 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
Feeding level
Bag material & pore size
60:40 hay:concentrate
Maintenance
Polyester, 40 -60 μm
Sample size: bag area 10-15 mg/cm 2
Particle size
No. of replicate animals > 2
Incubation sequence
Microbial correction
> 2 mm
All in, sequential removal
(with machine washing)
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
Correlation between selected buffer methods and in situ degradability
Method
Burroughs buffer
0.15M NaCl
Autoclaved rumen fluid
r
0.66
0.47
0.54

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 degradability
Bacterial
Bacteroides amyliphilus
Streptomyces griseus
Bacillus subtilis
Plant proteases
Papain & bromelain
Fungal proteases
Aspergillus oryzae
Animal proteases
Pancreatin & pepsin

In-situ versus protease degradability
R 2
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

14
C-Casein hydrolysis (mg/ml)
0.5 Co-culture
0.0 0.25
S. bovis
0.0
S. ruminantium
10 20
Time (h)
Single proteases can’t fully simulate microbial
activity of mixed rumen microbes

Degradability references
Broderick 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. CAB
International, Wallingford, pp. 233-254.
Huntington, J.A. and Givens, D.I., 1995. The in situ technique for studying the
rumen degradation of feeds: A review of the procedure. Nutrition Abstracts
and Reviews (Series B), 65: 65-93.
Orskov, E.R., 2000. The in situ technique for the estimation of forage
degradability. 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 bag
technique for the evaluation of feedstuffs. Tropical Animal Production, 5:
195-213.
Adesogan, A. T. Givens, D. I, and Owen, E. 2000. Chemical composition and
Nutritive Value of Forages. Field and Laboratory Methods for Grassland
and Animal Production Research (eds L t’Mannetje and R M Jones) pp 263-
278. CABI Publishing. Wallingford UK