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How to evaluate protein digestion in the ruminant? - SLU

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How to evaluate protein digestion in
the ruminant?
Peter UdГ©n
Kungsängen Research Centre, Swedish
University of Agricultural Sciences
Faculty of Veterinary Medicin and Animal Science
Department of Animal Nutrition and Management
Content
•
•
•
•
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Concepts of degradation
Protein fractions
Modeling microbial degradation of protein
Methods for evaluating ruminal degradation
Total tract indigestible protein
Concepts of degradation
• To pass a filter (insoluble protein): in vitro and in sacco
methods
• Molecular weight change (soluble proteins):
a) Electrophoretic methods – soluble protein degradation to
(at best) Mw<10 kDa
b) Precipitation methods – soluble protein degradation to (at
best) Mw<1 kDa
• The substance is metabolizable by the microorganisms: the
inhibited in vitro system – involves both extracellular
degradation, transport across the bacterial cell wall and intracellular metabolism of amino acids and peptides
Solubility of CP
Feed soures
Oats
A
B
C
D
E
F
BMM
NaCl
268
368
413
351
92
175
398
217
67
Solvent
MCD
BP
489
662
554
BCP
DDW
ARF
133
185
790
520
115
155
258
Wheat
B
217
256
D
436
614
E
F
G
Soybean
meal
A
C
D
F
G
Timothy
hay
A
C
G
403
292
208
462
406
297
370
197
193
241
130
130
215
279
208
140
63
113
152
198
BMM=Burroughs mineral mixture
diluted to 10% with distilled water
NaCl=sodium chloride solution
(0.5-0.9%)
MCD=McDougall’s artificial saliva
BP=borate-phosphate buffer
BCP=bicarbonate-phosphate
buffer
DDW, distilled deionised water
ARF=autoclaved rumen fluid.
169
295
245
276
236
274
354
274
320
222
236
Hedqvist, 2004
Distribution of N in forages
Feeds
Total N
BSN
c
Protein-Na
Protein-Nb
g/kg DM g/kg N
Fresh herbage
Ryegrass
White clover
Red clover
Birdsfoot trefoil Norcen
Birdsfoot trefoil Georgia One
Peptide-N
AA-N
NH3-N
g/kg BSN
23
40
34
24
23
372
361
390
352
249
363
238
189
386
210
221
152
115
196
173
396
325
325
446
420
96
113
106
93
112
7
27
18
7
6
Ryegrassd
21
668
13
-
-
367
93
White cloverd
41
509
34
-
-
380
126
Red cloverd
30
510
71
-
-
348
145
Birdsfoot trefoil Norcene
25
675
0
29
39
535
116
Birdsfoot trefoil Georgia Onee
25
566
14
38
30
510
146
Maize
13
529
13
Silage
aMethod
1 (Kjeldahl); bMethod 2 (ninhydrine)
Hedqvist, 2004
Modeling protein degradation
Casein degradation in vitro
Complex model
XC_AA
Prot
NH3_uptake
Simple model
NH3
AA _uptake
AA _exc h
Pr ot_P EP
Protein
End_p
PEP
IC_AA
Deam in
Degradation
PE P_u ptak e
UdГ©n, 1999
Casein degradation in vitro
Model
Flux
Complex model Proteolysis
Simple model
Direct fit
PEP uptake
AA uptake
AA exchange
Deamination
NH3 uptake
Proteolysis
Log fit
aPassage
= 0.166/h
k
(h-1)
km
( mol*g-1)
Vmax
( mol*g-1*h-1)
42
770
14
720
Escape
of AA (%)b
1.46
0.88
17
0.40
26
1.2
12
0.85
16
Data from Broderick & Craig, 1989
Casein simple model: non-linear fit
Casein
Rank 11 Eqn 8098 [Decay1_] y=aexp(-bx)
r^2=0.95910034 DF Adj r^2=0.91820068 FitStdErr=137.36116 Fstat=70.350239
a=1667.145
b=1.2037188
2000
1750
1500
Casein
1250
1000
750
500
250
0
0
0.5
1
Time (h)
1.5
2
Casein simple model: log-fit
Casein
Rank 1 Eqn 8160 [Line Robust None, Gaussian Errors] y=a+bx
r^2=0.91595425 DF Adj r^2=0.83190849 FitStdErr=0.23486937 Fstat=32.694843
a=7.2192237
b=-0.84936823
8
Casein
7.5
7
6.5
6
5.5
0
0.5
1
Time (h)
1.5
2
Complex model: casein and peptides
2,000
1
2
1,500
1
1,000
2
1
3
24
3
34
4
34
3
500
1
2
2
2
1
4
03
0.0
1
0.5
1.0
Time
1.5
2.0
Mod_casein
Obs_casein
Obs_XC_PEP
PEP
Methods for evaluating ruminal
degradation
• Buffer solubility
• Enzymatic methods
• In sacco methods
Methods for evaluating ruminal
degradation
• In vitro methods:
a) precipitation of remaining soluble proteins
b) electrophoretic identification of remaining
soluble proteins
c) inhibition of microbial synthesis –
measurement of NH3 + amino acids
d) NH3 – gas production method
e) Correction or removal of microbial
contamination of the feed protein
Precipitation:
in vitro incubation of whole rumen content with
soluble proteins
0,8
0,6
0,4
0,2
0,0
0
500
1000
1500
Dose mg TCA-N/kg RC
2000
Recovery of TCA-N after 1 min
Recovery of TCA-N after 1 min
Egg protein
Casein
1,0
1,0
0,8
0,6
0,4
0,2
0,0
0
1000
2000
3000
4000
Dose mg TCA-N/kg RC
Precipitation:
in vitro incubation of whole rumen content with casein
2,00
Casein degradation rate (/h)
1,80
1,60
1,40
1,20
1,00
0,80
0,60
0,40
0,20
0,00
0,00
2,00
4,00
6,00
8,00
10,00
12,00
Casein N dose (g/ca 6 kg rumen content)
14,00
Molecular weight (kDa)
Electrophoresis:
pea albumin proteins after in vitro incubation
Time (h)
Spencer et al.,
1988
Electrophoresis:
sunflower albumin 8 protein in vitro
McNabb et al., 1994
The inhibited in vitro method
The major problems:
• Dying culture – reduces maximum incubation time
and problems of estimating slow degrading proteins
• Hydrazine is now classified as a health hazard
The major advantages:
• Both for soluble and insoluble proteins
• Microbial contamination is no issue
The gas-in vitro method of Raab et al
The major problems:
• Ammonia from bacterial degradation – an issue
of protozoa, carbohydrates and bacterial lyses?
• Large no of flasks
The major advantages:
• Both for soluble and insoluble proteins
• Microbial contamination is no issue
Blanks in cubated with 3 levels of carbohydrates
16,00
Ammonia N (mg/flask)
14,00
12,00
10,00
8,00
6,00
4,00
2,00
0,00
0,00
-2,00
5,00
10,00
15,00
Time (h)
20,00
25,00
Blanks
- ammonia and gas production at 3 carbohydrate levels
16,00
14,00
12,00
23 h
1h
NH3-N
10,00
2
3.5
1h
8,00
4.5
6
6,00
7.5
10.5
4,00
17.5
23
2,00
0,00
0
-2,00
20
40
60
80
100
Gas production
120
140
160
Removal of microbial contamination of
the feed protein
Studies of the in vitro degradation of NDIN
• Washing, homogenisation, stomaching and
centrifugation results in losses of NDIN
• There is an accumulation of NDIN during in
vitro fermentation
Silke and UdГ©n, 2006 and unpubl.
NDIN remaining after 0 to 48 h fermentation in vitro
в—‹ fermented in vitro
fermented in vitro and washed in polyester bags
Silke and UdГ©n, unpubl.
Indigestible N = ADIN?
• Probably not
Recovery of ADIN
after 48-h in vitro fermentation
SAMPLE ID
%RECOVERY
ALFALFA HAY
20
ALF SILAGE 1
20
ALF SILAGE 2
27
ALF SILAGE 3
52
CORN SILAGE A
65
CORN SILAGE B
108
CORN SILAGE C
66
CORN SILAGE D
75
CORN SILAGE E
72
GRASS SILAGE
78
S-AMINE
9
S-HM
32
S-SBM
8
S-SOY
28
HAYCROP SILAGE
79
DISTIL GRAINS
54
AVERAGE
50
Robertson, unpubl.
Sheep digestion study with distillers
dried grains and solubles (DDGS)
• Eight 6-month old male sheep
• High quality grass silage and 4 levels of DDGS
wheat pellets: 0, 200, 400 and 600 g/kg DM
• Total collection of faeces and urine
Lucas plot of NDFIN (paper filtration)
Digested (g/kg DM)
6
5
4
y = 0.81x - 1.2615
RВІ = 0.989
3
2
1
0
-1
0
2
4
6
8
10
Consumed NDFIN (g/kg DM)
27
Digested (g/kg DM)
Lucas plot of ADFIN (paper filtration)
3
2.5
2
1.5
1
0.5
0
-0.5 0
y = 0.6645x - 0.561
RВІ = 0.9907
2
4
Consumed ADFIN (g/kg DM)
6
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