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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 :.

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Techniques for Measuring Feed Protein Digestion and Microbial Protein Synthesis

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  1. Techniques for Measuring Feed Protein Digestion and Microbial Protein Synthesis

  2. Laboratory estimates of protein degradability • Solubility in buffer and detergents • Incubation in controlled artificial rumen fermenter • Incubation with proteolytic enzymes

  3. 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

  4. 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

  5. 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

  6. 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

  7. The degradation rate(kd) kd = (ln B4 –ln B0 )/4 h. ruminal CP escape B0(kp /(kd +kp )) + C

  8. 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

  9. 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

  10. 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

  11. TABLE 1. Composition and estimated digestibilities of animal by-product .

  12. 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

  13. 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.

  14. Nylon pose / ”In situ” - metode

  15. 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 retrieve as group • Upon removal, wash bags under cold 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

  16. In situ : Dacronbags, 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

  17. 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)

  18. 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

  19. 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

  20. 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

  21. Interpretation of Results from Nylon Bags 100 80 Slowly digestible ‘b’ fraction Rate constant ‘c’ 60 CP Disappearance, % 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

  22. In situ ruminal degradation of crude protein in canola meal (CM), corn gluten meal (CGM) and fishmeal (FM) CM 100 80 FM 60 CP Disappearance, % CGM 40 20 0 0 12 24 36 48 60 72 Time of incubation, h

  23. 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

  24. 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 Effective degradability, % 40 CGM 20 .02 .04 .06 .08 .10 Fractional outflow rate, /h

  25. Problems with nylon bags • Standardising rumen liquor ?? • Micro-environments within bags • Particle loss from the bags • Contamination of residues with microbial matter

  26. Particle loss

  27. 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).

  28. Internal and external markers for quantifying microbial protein synthesis in the rumen Microbial fraction estimated Internal 2,6-Diaminopimelic acid (DAPA) Bacteria D-Alanine Bacteria 2-Aminoethylphosphonic acid (AEP) Protozoa Phosphatidyl choline Protozoa ATP Bacteria and protozoa Nucleic acids Bacteria and protozoa DNA RNA Individual purines and pyrimidines Total purines Nucleotide probes Bacteria and protozoa External 15N Bacteria and protozoa 35S Bacteria and protozoa 32P Bacteria and protozoa  

  29. 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

  30. Experimental timeline for protein digestibility study Days 7 0 14 21 26 Feed intake Dietary adaptation (14 d) Marker administration Microbial (15N) Digestibility (Yb) 0.42 0.41 0.40 0.39 0.38 15N enrichment of bacteria, atom % 0.37 0.36 Duodenal digesta Feces Rumen bacteria 0.35 0 2 4 6 10 12 8 15N infusion, d

  31. 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 OM flow(kg) - Microbial OM flow (kg))

  32. 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%

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