Techniques for Measuring Feed Protein Digestion and Microbial Protein Synthesis.

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 3°C

In vitro :

•Duplicate samples are incubated for 0 and 4 h at 3°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 °C for 15 min)

•supernatant fractions are stored at °C

•supernatant fractions analyzed for NH3 and TAAby a semiautomated method

In vitro :

•Degraded CP fraction (A0) , defined as the proportionof 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 6°C forced air oven

• weighed into 200-ml teflon-capped jars

Pepsin·HCl

• Fresh prewarmed (42 to 4°C) pepsin solution is added to each jar

•Jars are laid in a 4°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 6°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-mmWhole 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 rumenInsert at specific time intervals and

retrieve as groupUpon removal, wash bags under cold

water

•Incubation times 0 to 6 h: 3 to 6 time points6 to 24 h: 3 to 6 time points> 25 h: 6 to 12-h intervals

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 infor 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 minand were washed with the other bags to estimate thesoluble (degraded) CP fraction (A).

In situ incubations were replicated three times (twice in one cow and once in the other)

Bags were dried for 48 h at 6°C and weighedthen placed into a Kjeldahl flask for CP analysis

In situ :

Recommended guidelines for ruminal in situ degradation procedures

•Zero hour bags Incubate in artificial rumen fluid at 39°C for 30 min

•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 12 24 % error

Corn 0 4.8 3.6

Barley 3.8 22.4 3.8

Canola meal 1.8 3.9 .9

Soybean meal 14 19 15

Barley straw 165 146 205

Alfalfa hay 25 22 44

Percentage error = (|corrected N - uncorrected N|/corrected N) 100

Interpretation of Results from Nylon Bags

0

20

40

60

80

100

CP

Dis

appe

aran

ce,

%

0 12 24 36 48

Time of incubation, h

Soluble ‘a’ fraction

Slowly digestible ‘b’ fraction

Rate constant ‘c’

Degradation is described by an exponential equation:

y = a + b(1-e-c(t-L)) for t > L

In situ ruminal degradation of crude protein in canola meal (CM), corn gluten meal (CGM) and fishmeal (FM)

0

20

40

60

80

100C

P D

isap

pea

ran

ce,

%

0 12 24 36 48 60 72Time of incubation, h

CM

FM

CGM

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

Effect of ruminal outflow rate on effective degradability of crude protein in canola meal (CM), corn gluten meal (CGM) and fishmeal (FM)

20

40

60

80E

ffect

ive

degr

ada

bilit

y, %

.02 .04 .06 .08 .10 Fractional outflow rate, /h

CM

FM

CGM

Problems with nylon bags

• Standardising rumen liquor ??

• Micro-environments within bags

• Particle loss from the bags

• Contamination of residues with

microbial matter

0

0,2

0,4

0,6

0,8

1

1,2

0 10 20 30 40 50 60

Incubation time (h)

Frac

tion

degr

aded

Measureddegradationprofile

Corrected forparticle loss

Particle loss

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 estimatedInternal

2,6-Diaminopimelic acid (DAPA) BacteriaD-Alanine Bacteria2-Aminoethylphosphonic acid (AEP) ProtozoaPhosphatidyl choline Protozoa

ATP Bacteria and protozoa Nucleic acids Bacteria and protozoa

DNARNAIndividual purines and pyrimidines

Total purinesNucleotide probes Bacteria and protozoa

External15N Bacteria and protozoa35S Bacteria and protozoa32P 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

Days 0 2621147

Feed intake

Dietary adaptation (14 d)

Marker administration Microbial (15N) Digestibility (Yb)

Duodenal digestaFecesRumen bacteria

Experimental timeline for protein digestibility study

0.35

0.36

0.37

0.38

0.39

0.40

0.41

0.42

15N

en

rich

me

nt

of

ba

cte

ria,

ato

m %

0 2 4 6 8 10 12

15N infusion, d

Protein digestion and microbial protein synthesis in a lactating dairy cow

Item Value Calculation

N intake, g/d 558 DM intake (kg/d) Feed N (g/kg)

Duodenal N flow

Total N

g/d 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)

% N intake 97.8 Duod N flow (g/d)/N intake (g/d) 100%

NH3-N, g/d 20.4

NAN

g/d 526 Total N flow (g/d) - NH3-N flow (g/d)

% N intake 94.2 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 OMflow(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 5.7 (N intake (g/d) - Duod NAN flow (g/d))/N intake (g/d) 100%

Corrected 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%