Transcript No Slide Title

Techniques for Measuring
Feed Protein Digestion and
Microbial Protein Synthesis
Laboratory estimates of protein degradability
• Solubility in buffer and detergents
• Incubation in controlled artificial rumen
fermenter
• Incubation with proteolytic enzymes
In vitro :
•Samples are ground (1-mm screen)
•weighed into duplicate 50-ml centrifuge tubes
•Five milliliters of McDougall’s buffer (14) are
added to each sample
•allowed to soak for 60 to 90 min at 39°C
In vitro :
•Duplicate samples are incubated for 0 and 4 h
at 39°C after addition of 10 ml of RF buffer
inoculum
•Inhibitor concentrations are 1.0 mM hydrazine and
30 mg of chloramphenicol/ml,which are added to
suppress microbial uptake of NH3 and TAA
•Incubations are stopped by the addition of 5%
(wt/vol) TCA and placement of the tubes on ice for
30 min
In vitro :
•samples are centrifuged (15,300* g at 4°C for 15 min)
•supernatant fractions are stored at 4°C
•supernatant fractions analyzed for NH3 and TAA
by a semiautomated method
In vitro :
•Degraded CP fraction (A0) , defined as the proportion
of total N present as NH3 and TAA at 0 h
•Potentially degradable CP fraction present at 0 h ( B0 )
was defined as 100 – A0
•CP fraction remaining undegradedat 4 h ( B4 )
was defined as 100 – A4
(A4 ): defined as the proportion of total N present
as NH3 and TAA at 4 h
The degradation rate(kd)
kd = (ln B4 –ln B0 )/4 h.
ruminal CP escape
B0(kp /(kd +kp )) + C
Pepsin·HCl
• Five grams (air-dried basis) of ground
sample are weighed in duplicate into folded
• placed into ether extraction cylinders; and extracted
for 72 h to remove lipid
• dried for 24 h in a 60°C forced air oven
• weighed into 200-ml teflon-capped jars
Pepsin·HCl
• Fresh prewarmed (42 to 45°C) pepsin solution
is added to each jar
•Jars are laid in a 45°C incubator-shaker for 16 h.
• After incubation allowed to sit for 15 min
• Residues are filtered
Pepsin·HCl
•Residues and filter papers are rinsed with acetone
• dry over-night in a 60°C forced-air oven
•transfer directly to Kjeldahl flasks
digestible CP = [1 – (residual CP/total CP)] * 100
TABLE 1. Composition and estimated digestibilities of animal by-product
.
In situ/In sacco Techniques
• In situ = In place
• In sacco = In bag
• Suspend a bag containing feed in rumen or
cecum
• Mobile nylon bag- placed into duodenum
and collected at ileum +/or feces
In situ nylon bag technique
(in sacco technique)
• Used to determine degradation of protein in
protein supplements and basal feeds.
• Requires rumen cannulated animals.
• Feedstuffs contained in bags made from
polyester (nylon) cloth are incubated in the rumen
for a range of times, and the degradation loss for
each incubation time is measured.
Nylon pose / ”In situ” - metode
Recommended guidelines for ruminal
in situ degradation procedures
•Bag porosity
40 to 60 m
•Particle size
Protein supplements, 2-mm
Whole grains, hays and silages, 5-mm
•Sample size to bag surface area
10 to 20 mg/cm2
•Pre-ruminal incubation
Soak bags in water/buffer prior to
incubation
•Bag insertion and removal
Weight bags to position in rumen
Insert at specific time intervals and
as group
Upon removal, wash bags under cold
retrieve
water
•Incubation times
0 to 6 h: 3 to 6 time points
6 to 24 h: 3 to 6 time points
> 25 h: 6 to 12-h intervals
In situ :
Dacron bags, 9 * 12 cm (52- mm pore size)
were filled with 2 g of ground (2-mm screen)
incubated in the ventral rumen of two cows in
for 4, 8, 12, 16, 20, 24,36, 48, 72, and 96 h
removal from the rumen, bags were immediately
soaked in ice water and transferred to a washing
machine for rinsing
In situ :
Zero-hour bags were soaked in tapid water for 30 min
and were washed with the other bags to estimate the
soluble (degraded) CP fraction (A).
Bags were dried for 48 h at 60°C and weighed
then placed into a Kjeldahl flask for CP analysis
In situ incubations were replicated three times
(twice in one cow and once in the other)
Recommended guidelines for ruminal
in situ degradation procedures
•Zero hour bags
min
Incubate in artificial rumen fluid at 39°C for 30
•Animal/period
Use type of animal for which the digestion rate
determinations are to be applied
Replicate
•Diet
Feed ingredients to be tested included in the
basal diet
•Microbial contamination Use of microbial marker to correct for
contamination
Especially for low quality forages
•The degredation rate of in vitro method
were higher than in situ method
• Linear regression indicated that degradation
rates estimated by IIV technique were highly
correlated with those estimated by the IS method
• All two procedures ranked the animal by product proteins
similarly for degradation rate and ruminal escape
• Of these two methods, the IIV method was the
most rapid and required the least labor
Effect of bacterial nitrogen contamination on the
percent error associated with determination of
residual nitrogen
Ruminal incubation time, h
Ingredient
5-6
Corn
Barley
Canola meal
Soybean meal
Barley straw
Alfalfa hay
0
3.8
1.8
14
165
25
12
% error
4.8
22.4
3.9
19
146
22
24
3.6
3.8
.9
15
205
44
Percentage error = (|corrected N - uncorrected N|/corrected N)  100
Interpretation of Results from Nylon Bags
CP Disappearance, %
100
80
Slowly
digestible
‘b’ fraction
Rate constant ‘c’
60
40
Soluble ‘a’
fraction
20
0
0
12
24
36
48
Time of incubation, h
Degradation is described by an exponential
equation:
y = a + b(1-e-c(t-L)) for t > L
CP Disappearance, %
In situ ruminal degradation of crude protein in canola
meal (CM), corn gluten meal (CGM) and fishmeal (FM)
100
CM
80
60
FM
40
CGM
20
0
0
12
24
36
48
60
Time of incubation, h
72
Effective degradability
•Effective degradability (ED) = a + b × c/(c + k)
where: a, b and c are constants as defined
previously
k = fractional outflow rate from the rumen (/h)
•Typically values for k:
0.02 to 0.10 for protein supplements
0.017 to 0.05 for forages
Effective degradability, %
Effect of ruminal outflow rate on effective
degradability of crude protein in canola meal (CM),
corn gluten meal (CGM) and fishmeal (FM)
80
CM
60
FM
40
CGM
20
.02
.04
.06
.08
Fractional outflow rate, /h
.10
Problems with nylon bags
• Standardising rumen liquor ??
• Micro-environments within bags
• Particle loss from the bags
• Contamination of residues with
microbial matter
Fraction degraded
1,2
Measured
degradation
profile
1
0,8
0,6
0,4
Corrected for
particle loss
Particle loss
0,2
0
0
10
20
30
40
Incubation time (h)
50
60
In vivo determination of protein digestion
and microbial protein synthesis
• Requires ruminally and abomasally or duodenally
(anterior to the pancreatic and bile ducts) cannulated
animals.
• Differentiation between feed protein and microbial
protein flowing to the duodenum (use of microbial
markers).
Internal and external markers for quantifying microbial
protein synthesis in the rumen
Microbial fraction estimated
Internal
2,6-Diaminopimelic acid (DAPA)
D-Alanine
2-Aminoethylphosphonic acid (AEP)
Phosphatidyl choline
ATP
Nucleic acids
DNA
RNA
Individual purines and pyrimidines
 Total purines
Nucleotide probes
External
 15N
35S
32P
Bacteria
Bacteria
Protozoa
Protozoa
Bacteria and protozoa
Bacteria and protozoa
Bacteria and protozoa
Bacteria and protozoa
Bacteria and protozoa
Bacteria and protozoa
Microbial markers - cont’d
• Purine derivatives
– microbial nucleic acids are extensively degraded in the intestine
yielding purines
– microbial purines are absorbed and the majority are metabolized by
the animal to allantoin, uric acid, xanthine and hypoxanthine (in
sheep) and excreted in urine
– amount of microbial N reaching duodenum is calculated from the
excretion of purine derivatives in urine
– requires total collection of urine
Experimental timeline for protein digestibility study
7
0
14
21
26
Feed intake
enrichment of bacteria, atom %
Dietary adaptation (14 d)
15N
Days
Marker administration
Microbial (15N)
Digestibility (Yb)
0.42
0.41
0.40
0.39
0.38
0.37
0.36
0.35
0
2
4
15N
6
8
infusion, d
10
12
Duodenal digesta
Feces
Rumen bacteria
Protein digestion and microbial protein synthesis in a lactating dairy
cow
Item
N intake, g/d
Value
Calculation
558
DM intake (kg/d)  Feed N (g/kg)
546
Duod DM flow (kg/d)  Duod N (g/kg)
Duod DM flow (kg/d)= Intake of digestibility marker (g/d)/
Marker in duod digesta (g/kg)
Duodenal N flow
Total N
g/d
% N intake
NH3-N, g/d
97.8
Duod N flow (g/d)/N intake (g/d)  100%
20.4
NAN
g/d
% N intake
526
94.2
Total N flow (g/d) - NH3-N flow (g/d)
NAN flow (g/d)/N intake (g/d)  100%
Microbial N
g/d
286
Duod marker flow (g/d)/
(Microbial marker/Microbial N (g/d) )
% of NAN
54.4
Microbial N flow (g/d)/NAN flow (g/d)  100%
g/kg RFOM
23.4
Microbial N flow (g/d)/((OM intake (kg) - Duod OM
flow(kg) - Microbial OM flow (kg))
Protein digestion and microbial protein synthesis in a lactating dairy
cow -cont’d
Item
Value
Calculation
Duodenal N flow
Feed N
g/d
240
Total N flow (g/d) - Microbial N flow (g/d) NH3-N flow (g/d)
% NAN
45.6
Feed N flow (g/d)/ NAN flow (g/d)  100%
% N intake
44
Feed N flow (g/d)/N intake (g/d)  100%
Digestibility, %
Ruminal
Apparent
Corrected
5.7
(N intake (g/d) - Duod NAN flow (g/d))/
N intake (g/d)  100%
57
((N intake (g/d) - (Duod NAN flow (g/d) Microbial Nflow (g/d)))/N intake (g/d)  100%
Post-ruminal
72.2
(Duod NAN flow (g/d) - Fecal N (g/d))/
Duod NAN flow (g/d)  100%
Total tract
73.8
(N intake (g/d) - Fecal N (g/d))/N intake (g/d) 
100%