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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