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Techniques for Measuring Feed Protein Digestion and Microbial Protein Synthesis.

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Presentation on theme: "Techniques for Measuring Feed Protein Digestion and Microbial Protein Synthesis."— Presentation transcript:

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 3  °C

4 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

5 In vitro : samples are centrifuged (15,300*  g at  °C for 15 min) supernatant fractions are stored at  °C supernatant fractions analyzed for NH 3 and TAA by a semiautomated method

6 In vitro : Degraded CP fraction (A 0 ), defined as the proportion of total N present as NH3 and TAA at 0 h Potentially degradable CP fraction present at 0 h ( B 0 ) was defined as 100 – A 0 CP fraction remaining undegradedat 4 h ( B 4 ) was defined as 100 – A 4 (A 4 ): defined as the proportion of total N present as NH3 and TAA at 4 h

7 The degradation rate(k d ) k d = (ln B 4 –ln B 0 )/4 h. ruminal CP escape B 0 (k p /(k d +k p )) + 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 6  °C forced air oven weighed into 200-ml teflon-capped jars

9 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

10 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

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



17 Recommended guidelines for ruminal in situ degradation procedures Bag porosity40 to 60  m Particle sizeProtein supplements, 2-mm Whole grains, hays and silages, 5-mm Sample size to bag surface area10 to 20 mg/cm 2 Pre-ruminal incubationSoak bags in water/buffer prior to incubation Bag insertion and removalWeight bags to position in rumen Insert at specific time intervals and retrieve as group Upon removal, wash bags under cold water Incubation times0 to 6 h: 3 to 6 time points 6 to 24 h: 3 to 6 time points > 25 h: 6 to 12-h intervals

18 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 :

19 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). 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 weighed then placed into a Kjeldahl flask for CP analysis In situ :

20 Recommended guidelines for ruminal in situ degradation procedures Zero hour bagsIncubate in artificial rumen fluid at 39°C for 30 min Animal/periodUse type of animal for which the digestion rate determinations are to be applied Replicate DietFeed ingredients to be tested included in the basal diet Microbial contaminationUse of microbial marker to correct for contamination Especially for low quality forages



23 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

24 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 straw165146205 Alfalfa hay 25 22 44 Percentage error = (|corrected N - uncorrected N|/corrected N)  100

25 Interpretation of Results from Nylon Bags 0 20 40 60 80 100 CP Disappearance, % 012243648 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

26 In situ ruminal degradation of crude protein in canola meal (CM), corn gluten meal (CGM) and fishmeal (FM) 0 20 40 60 80 100 CP Disappearance, % 0122436486072 Time of incubation, h CM FM CGM

27 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

28 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 80 Effective degradability, %. Fractional outflow rate, /h CM FM CGM

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


31 Particle loss

32 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).

33 Internal and external markers for quantifying microbial protein synthesis in the rumen Microbial fraction estimated Internal 2,6-Diaminopimelic acid (DAPA)Bacteria D-AlanineBacteria 2-Aminoethylphosphonic acid (AEP)Protozoa Phosphatidyl cholineProtozoa ATPBacteria and protozoa Nucleic acidsBacteria and protozoa DNA RNA Individual purines and pyrimidines Total purines Nucleotide probesBacteria and protozoa External 15 NBacteria and protozoa 35 SBacteria and protozoa 32 PBacteria and protozoa  

34 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

35 Days 0262114 7 Feed intake Dietary adaptation (14 d) Marker administration Microbial ( 15 N) Digestibility (Yb) Duodenal digesta Feces Rumen bacteria Experimental timeline for protein digestibility study 0.35 0.36 0.37 0.38 0.39 0.40 0.41 0.42 15 N enrichment of bacteria, atom % 0246 8 1012 15 N infusion, d

36 Protein digestion and microbial protein synthesis in a lactating dairy cow Item ValueCalculation N intake, g/d558DM intake (kg/d)  Feed N (g/kg) Duodenal N flow Total N g/d546Duod 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 intake97.8Duod N flow (g/d)/N intake (g/d)  100% NH 3 -N, g/d20.4 NAN g/d526Total N flow (g/d) - NH 3 -N flow (g/d) % N intake94.2NAN flow (g/d)/N intake (g/d)  100% Microbial N g/d286Duod marker flow (g/d)/ (Microbial marker/Microbial N (g/d) ) % of NAN54.4Microbial N flow (g/d)/NAN flow (g/d)  100% g/kg RFOM23.4Microbial N flow (g/d)/((OM intake (kg) - Duod OM flow(kg) - Microbial OM flow (kg))

37 Protein digestion and microbial protein synthesis in a lactating dairy cow -cont’d Item ValueCalculation Duodenal N flow Feed N g/d240Total N flow (g/d) - Microbial N flow (g/d) - NH 3 -N flow (g/d) % NAN45.6Feed N flow (g/d)/ NAN flow (g/d)  100% % N intake44Feed N flow (g/d)/N intake (g/d)  100% Digestibility, % Ruminal Apparent5.7(N intake (g/d) - Duod NAN flow (g/d))/ N intake (g/d)  100% Corrected57((N intake (g/d) - (Duod NAN flow (g/d) - Microbial Nflow (g/d)))/N intake (g/d)  100% Post-ruminal72.2(Duod NAN flow (g/d) - Fecal N (g/d))/ Duod NAN flow (g/d)  100% Total tract73.8(N intake (g/d) - Fecal N (g/d))/N intake (g/d)  100%

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