Presentation on theme: "Techniques for Measuring Feed Protein Digestion and Microbial Protein Synthesis To establish the amounts and ratios of nutrients necessary for optimal."— Presentation transcript:
1Techniques for Measuring Feed Protein Digestion and Microbial Protein Synthesis To establish the amounts and ratios of nutrients necessary for optimal microbial and animal response, one must first adequately predict the degree to which nutrients are made available in the rumen from a variety of dietary ingredients
2Laboratory estimates of protein degradability Solubility in buffer and detergentsIncubation in controlled artificial rumen fermenterIncubation with proteolytic enzymes
3In vitro :Samples are ground (1-mm screen)weighed into duplicate 50-ml centrifuge tubesFive milliliters of McDougall’s buffer (14) are added to each sampleallowed to soak for 60 to 90 min at 39°C
4In vitro :Duplicate samples are incubated for 0 and 4 h at 39°C after addition of 10 ml of RF buffer inoculumInhibitor concentrations are 1.0 mM hydrazine and30 mg of chloramphenicol/ml,which are added tosuppress microbial uptake of NH3 and TAAIncubations are stopped by the addition of 5% (wt/vol) TCA and placement of the tubes on ice for 30 min
5In vitro :samples are centrifuged (15,300* g at 4°C for 15 min)supernatant fractions are stored at 4°Csupernatant fractions analyzed for NH3 and TAAby a semiautomated method
6In vitro :Degraded CP fraction (A0) , defined as the proportionof total N present as NH3 and TAA at 0 hPotentially degradable CP fraction present at 0 h ( B0 )was defined as 100 – A0CP fraction remaining undegradedat 4 h ( B4 )was defined as 100 – A4(A4 ): defined as the proportion of total N presentas NH3 and TAA at 4 h
8Pepsin·HClFive grams (air-dried basis) of ground sample are weighed in duplicate into foldedplaced into ether extraction cylinders; and extractedfor 72 h to remove lipiddried for 24 h in a 60°C forced air ovenweighed into 200-ml teflon-capped jars
9Pepsin·HClFresh prewarmed (42 to 45°C) pepsin solutionis added to each jarJars are laid in a 45°C incubator-shaker for 16 h.After incubation allowed to sit for 15 minResidues are filtered
10Pepsin·HClResidues and filter papers are rinsed with acetonedry over-night in a 60°C forced-air oventransfer directly to Kjeldahl flasksdigestible CP = [1 – (residual CP/total CP)] * 100
11TABLE 1. Composition and estimated digestibilities of animal by-product
12In situ/In sacco Techniques In situ = In placeIn sacco = In bagSuspend a bag containing feed in rumen or cecumMobile nylon bag- placed into duodenum and collected at ileum +/or feces
13In 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.The in situ nylon bag technique allows intimate contact of the test feed with the rumen environment including temperature, pH, buffer, substrate, enzymes, although when incubated within the rumen, the feed is not subject to the total rumen experience (mastication, rumination, and passage).
17Recommended guidelines for ruminal in situ degradation procedures Bag porosity 40 to 60 mParticle size Protein supplements, 2-mmWhole grains, hays and silages, 5-mmSample size to bag surface area 10 to 20 mg/cm2Pre-ruminal incubation Soak bags in water/buffer prior to incubationBag insertion and removal Weight bags to position in rumenInsert at specific time intervals and retrieve as groupUpon removal, wash bags under cold waterIncubation times 0 to 6 h: 3 to 6 time points6 to 24 h: 3 to 6 time points> 25 h: 6 to 12-h intervalsBag porosity (pore size): is a compromise between allowing influx of microbial populations to degrade the test feed and escape of accumulated gases, while at the same time minimizing the influx of rumen contents not associated with the test feed and the efflux of undegraded feed particles.Sample size to bag surface area: optimum sample size is that which provides enough residue at the end of extended rumen incubation for chemical analysis without over filling the bag so as to delay bacterial attachment, increase lag time, and underestimate digestion rates.10 to 20 mg/cm2: if using 10 × 15 cm bags then the surface area is equal to 10 × 15 × 2 sides = 300 cm2 , therefore 3 to 6 g of sample are used.Incubation times: bags are incubated and withdrawn at various times so that a description of degradation over time is obtained. The number of time points during the digestion sequence should be adequate to detect an observable lag time (1-h intervals) and an end-point of digestion. For most protein supplements and concentrate ingredients, 48 to 72 h of incubation is adequate to detect a ruminal digestion end-point. For forages, 72 to 108 h of incubation may be required.
18In 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 infor 4, 8, 12, 16, 20, 24,36, 48, 72, and 96 hremoval from the rumen, bags were immediatelysoaked in ice water and transferred to a washing machine for rinsing
19In 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).Bags were dried for 48 h at 60°C and weighedthen placed into a Kjeldahl flask for CP analysisIn situ incubations were replicated three times (twice in one cow and once in the other)
20Recommended guidelines for ruminal in situ degradation procedures Zero hour bags Incubate in artificial rumen fluid at 39°C for 30 minAnimal/period Use type of animal for which the digestion rate determinations are to be appliedReplicateDiet Feed ingredients to be tested included in thebasal dietMicrobial contamination Use of microbial marker to correct for contaminationEspecially for low quality forages
23The 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 methodAll two procedures ranked the animal by product proteinssimilarly for degradation rate and ruminal escapeOf these two methods, the IIV method was themost rapid and required the least labor
24Effect of bacterial nitrogen contamination on the percent error associated with determination of residual nitrogenRuminal incubation time, hIngredient% errorCornBarleyCanola mealSoybean mealBarley strawAlfalfa hayPercentage error = (|corrected N - uncorrected N|/corrected N) 100Concentrate ingredients generally contain little microbial contamination (5 to 10% of residual N), except for barley which can have quite high microbial contamination at 12 h incubation. Forages with low N content and slow rates of degradability tend to have more contamination of the residue. When forages are corrected for bacterial N contamination they will have reduced digestion, reduced lag times less nondigestible residue, and faster N digestion rates.Low protein forages and coarse feedstuffs should be corrected for microbial contamination.
25Interpretation of Results from Nylon Bags 10080Slowly digestible ‘b’ fractionRate constant ‘c’60CP Disappearance, %40Soluble ‘a’ fraction2012243648The complete degradation curve is sigmoid in shape. Most of the curve is described by an exponential equation:y = a + b(1-e-c(t-L)), for t > Lwhere: y = CP degradation at time t (%)t = time of incubaton (hours)a = the rapidly soluble fraction (%)b = the fraction that will degraded in the rumen in time, the slowly digestible fraction (%)c = the fractional rate of disappearance; the rate at which fraction b will be degraded per hour (%/h)The sum of fraction a and b is equal to the potentially degradable fraction. It follows, therefore, that (a + b) is the percentage which is totally undegradable in the rumen.Time of incubation, hDegradation is described by an exponential equation:y = a + b(1-e-c(t-L)) for t > L
26In situ ruminal degradation of crude protein in canola meal (CM), corn gluten meal (CGM) and fishmeal (FM)100CM80FM60CP Disappearance, %40CGM20122436486072Time of incubation, h
27Effective degradability Effective degradability (ED) = a + b × c/(c + k)where: a, b and c are constants as defined previouslyk = fractional outflow rate from the rumen (/h)Typically values for k:0.02 to 0.10 for protein supplements0.017 to 0.05 for foragesThe degradation curves shown on the previous pages were obtained by retaining the samples in the rumen (by containment in a nylon bag). Normally food material can leave the rumen once its particle size has been reduced by degradation and rumination. Many concentrate foods and supplements (eg canola meal, fishmeal) are already of a particle size small enough to leave the rumen without further size reduction. Thus the degradation actually achieved within the rumen, the effective degradation, will depend on how long the food remains within the rumen (i.e., the retention time).
28Effect of ruminal outflow rate on effective degradability of crude protein in canola meal (CM), corn gluten meal (CGM) and fishmeal (FM)80CM60FMEffective degradability, %40CGM20.02.04.06.08.10Fractional outflow rate, /h
29Problems with nylon bags Standardising rumen liquor ??Micro-environments within bagsParticle loss from the bagsContamination of residues with microbial matter
32In 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).
33Microbial fraction estimated Internal and external markers for quantifying microbial protein synthesis in the rumenMicrobial fraction estimatedInternal2,6-Diaminopimelic acid (DAPA) BacteriaD-Alanine Bacteria2-Aminoethylphosphonic acid (AEP) ProtozoaPhosphatidyl choline ProtozoaATP Bacteria and protozoa Nucleic acids Bacteria and protozoaDNARNAIndividual purines and pyrimidinesTotal purinesNucleotide probes Bacteria and protozoaExternal15N Bacteria and protozoa35S Bacteria and protozoa32P Bacteria and protozoaMicrobial markers may be classified as internal markers (inherently present in microorganisms) and external markers (markers added to the rumen to label the microorganisms).DAPA (D-Alanine)- located in cell wall- overestimates bacterial nitrogen flow when lysis of bacterial cells is large. Intraruminal degradation of bacterial protoplasmic proteins is more rapid and extensive than cell-wall residues and their constituent DAP, thus total DAP flowing out of the rumen is disproportionately highAEP- widespread distribution of AEP and other aminophosphonic acids among ruminal bacteria and presence in feedstuffsATP- rapid hydrolysis of ATP and little or no ATP formation in inactive or dead cellsNucleic acids- DNA and RNA unstable- presence of nucleic acids in feedstuffs but content is low and degradation in rumen is nearly complete- may overestimate microbial N flow with feedstuffs of low rumen degradability, particularly at high ruminal outflow rates15N - stable isotope- most reliable- introduced into the rumen as 15N-ammonium salts [(15NH4)2SO4, 15NH4Cl]- bacterial N is labelled by direct incorporation of 15N-ammonia35S and 32P - radioactive isotopes- 35S incorporated into cyst and met and other S-containing compounds- 32P incorporated into phospholipids
34Microbial markers - cont’d Purine derivativesmicrobial nucleic acids are extensively degraded in the intestine yielding purinesmicrobial purines are absorbed and the majority are metabolized by the animal to allantoin, uric acid, xanthine and hypoxanthine (in sheep) and excreted in urineamount of microbial N reaching duodenum is calculated from the excretion of purine derivatives in urinerequires total collection of urine
35Experimental timeline for protein digestibility study Days7142126Feed intakeDietary adaptation (14 d)Marker administrationMicrobial (15N)Digestibility (Yb)0.420.410.400.3915N enrichment of bacteria, atom %0.380.370.36Duodenal digestaFecesRumen bacteria0.352468101215N infusion, d
36Protein digestion and microbial protein synthesis in a lactating dairy cow Item Value CalculationN intake, g/d 558 DM intake (kg/d) Feed N (g/kg)Duodenal N flowTotal Ng/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 20.4NANg/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 Ng/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))
37Protein digestion and microbial protein synthesis in a lactating dairy cow -cont’d Item Value CalculationDuodenal N flowFeed Ng/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, %RuminalApparent 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%