In vitro Gas Production Technique

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WELCOME

Presented by:- Tapan Kumar Das Ph.D. Scholar DCN division : 

Presented by:- Tapan Kumar Das Ph.D. Scholar DCN division Scope and Improvement of In Vitro Gas Production Technique in Feed Evaluation AN-619

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Introduction ?The nutritive value or energy content of an animal feed is determined predominately by its digestibility. ?In vivo studies are not suited for the rapid and routine feed evaluations undertaken by commercial laboratories. ?In vitro techniques which are conducted outside of the animal system but simulate the digestion process.

Tilley and Terry method (1963) : 

Tilley and Terry method (1963) ?End-point measurement (gives only one observation). (Makkar et al., 1993) ?Overestimation of DMD for tannin-rich feeds. ?Does not provide information on the kinetics of forage digestion.

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In vitro gas Production technique (IVGPT) (Menke et al., 1979)

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Rumen fluid collection & preparation Syringe & Medium preparation and maintained at 390c Filling of syringe Initial recording Incubate the syringe Addition of rumen fluid into the medium Procedure Final reading Degradability, CH4, NH3, VFA Weigh feed sample

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Advantages of IVGPT ?More efficient than the in sacco method in evaluating the effects of tannins or other anti-nutritive factors. ? Needs simple equipment. ?Focuses on the end products of fermentation and not DM disappearance (Tilley-Terry; in situ). ?Small amount of sample required. ?Digestion kinetic studies.

Factors affecting accuracy of in-vitro fermentation : 

Factors affecting accuracy of in-vitro fermentation ?Sample form ?Oven-drying samples ?Buffer composition ?Inoculum to buffer ratio ?Prevailing pH and temperature ?Stirring

Application : 

Application

Measuring of Methane production : 

Measuring of Methane production After finishing of incubation a sample total gas volume should be taken and injected to the GC. CH4 can be measured by following formula: CH4=(area of standard × area of sample)/area of standard 1. Use of Gas Chromatograph:- (Getachew et al., 2005 )

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NaOH can absorb CO2 content of syringe so only methane remains and it can calculated by: CH4 (ml) = Total gas volume after incubation – gas volume after injection of NaOH 2. Use of NaOH (10M): ? It can be used in those laboratories which GC equipments are not available. ?Another advantages is that it is very simple and inexpensive. (Fievez et al., 2005)

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3. Stoichiometrically calculation of methane from VFA production:- (Blümmel et al., 1997 and Getachew et al., 1998) Fermentative CO2 = (A/2) + (P/4) + 1.5B (where A =Moles of acetate P =Moles of propionate B =Moles of butyrate) Fermentative CH4 = (A+2B)-CO2 (where A =Moles of acetate B=Moles of butyrate CO2 =Moles of CO2 calculated from previous equation.)

Methane measured (24 hr.) and Stoichiometrically calculated from VFA production : 

Methane measured (24 hr.) and Stoichiometrically calculated from VFA production (Getachew et al., 2005)

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Estimation of SCFA Prediction of SCFA from in vitro gas measurement will be increasingly important in developing countries where laboratories are seldom equipped with modern equipments to measure SCFA. mmol SCFA=0.0222(ml gas at 24h)-0.00425 (Getachew et al., 2002)

Determination of Energy Content of Feeds : 

Determination of Energy Content of Feeds ?Prediction of ME is more accurate when based on gas and chemical constituents measurement as compared to calculation based on chemical constituents only. * ? IVGPT has been widely used to evaluate ME energy value of several classes of feeds. (Krishnamoorthy et al., 1995) ?It has been reported that there is a close relationship between the energy value of feed calculated on in vivo digestion trial and in vitro gas production methods. (Menke & Steingass, 1988) (Menke et al., 1979)

Where, GP = 24-h net gas production (ml/200 mg DM) CA = crude ash (% DM) : 

Where, GP = 24-h net gas production (ml/200 mg DM) CA = crude ash (% DM) Calculation of ME and NEL values in feeds using equations of Menke and Steingass (1988) ME (MJ/kg DM)= 0.157*GP+0.0084*CP+0.022*EE- 0.0081*CA+1.06 For concentrate, grains and by-products:- NEL (MJ/kg DM)= 0.096*GP+0.0038*CP+ 0.000173* EE2 + 0.54 NEL (MJ/kg DM)= 0.115*GP+0.0054*CP+0.014*EE- 0.0054*CA- 0.36 For forages: ME (MJ/kg DM)= 0.136*GP+0.0057*CP+ 0.000286*EE2 + 2.20

Kinetics of rumen fermentation : 

Kinetics of rumen fermentation Allows recording of gas produced at several times which is used to predict the rate at which feed is digested. The gas method has been used to evaluate the effects of grain processing on the rate and extent of gas production. (DePeters et al., 2003) Kinetics of in vitro gas production during incubation of flaked and whole corn grain.

Prediction of Voluntary Feed Intake : 

Prediction of Voluntary Feed Intake ?IVGPT has been used to predict DMI, because there was significant correlation between in vitro gas production and DMI. ( Blümmel et al., 1997) ?Forages with high voluntary DMI have high gas volumes at early incubation times (i.e., 2-8 h) and high substrate true degradabilities but low gas production at later times of incubation. ( Blümmel et al., 2005) *

Evaluation of Anti-nutritive Factors : 

Evaluation of Anti-nutritive Factors 3) To evaluate various commercially available chemicals which have affinity to tannins for their binding capacity of tannins such as polyethylene glycol (PEG). 1) To study the action of tannins, saponins and alkaloids. 2) To evaluate interaction that takes place during fermentation for example interaction between saponins and tannins. (Makkar, 2005)

Effect of added fat on Gas production : 

Effect of added fat on Gas production ?Unless included at relatively low level (<50g/kg DM) or in rumen protected form, lipid supplements adversely affect fibre degradation. ?Fatty acid in the form of triglyceride had no effect on gas production but in form of potassium salt significantly depress gas production (Getachew et al., 2001)

Effect of yellow grease (YG) and yellow grease soap (YG soap) on in vitro gas production : 

Effect of yellow grease (YG) and yellow grease soap (YG soap) on in vitro gas production YG-Yellow grease YG Soap- yellow grease soap (Getachew et al., 2001)

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?Positive associative effects on in vitro gas production occurred when rice straw was incubated in mixtures with hay or mulberry leaves. Associative effect ?Ingredient combination in mixed diets reported to have associative effect on gas production. (Getachew et al., 2005) (Liu et al., 2002)

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Cumulative gas produced at different times of incubation of Rice Straw (RS) with Ryegrass Hay (Hay) or Mulberry Leaves (ML) (Liu et al., 2002)

Efficiency of Microbial Biomass Production (EMBP) : 

Efficiency of Microbial Biomass Production (EMBP) ?Partitioning of degraded nutrients such that more microbial mass is produced at the cost of gas, and/or SCFA, production; reflecting higher microbial efficiency. ?This higher microbial efficiency can not be detected if only gas production is measured. ( Makkar, 2005) ?For measurement of TDOM of syringe contents with NDS is being used for conventional feeds but not in tannin-rich sample. ( Makkar, 2004)

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?15N incorporation from buffer containing 15N labeled ammonium bicarbonate or measurement of purines or diaminopimelic acid (DAPA) has to be used as an index of microbial protein production in studies where tannin-rich plants were incubated in an in vitro gas production technique. In this situation TDS can be estimate: Truly degraded substrate (mg)=total markers in syringe (mg) ×100/percent marker in rumen microbes. ( Makkar, 2005) cont….

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?Grings et al. (2005) tested relationship between in vivo and in vitro estimates of EMBP for silage-based diets. They concluded that EMBP estimated by in vitro gas production method was well related to in vivo measures of microbial efficiency. EMBP={TSD-(gas volume × SF)}/TSD TSD: True substrate digestibility SF: Stoichiometric factor =2.2 and 2.34 in case of high proportional propionate (>40 %) ( Blümmel, 2000) Microbial mass= TSD-(gas volume × SF) In vitro:

Limitation : 

Limitation

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Blank results should always be reported in table of results not subtracted from sample results. Blank do not produce gas at the same rate as the sample. Dynamic calculation is incorrect if cumulative volume (CV) of gas from blank is simply deducted from the CV of gas from the sample. (Williams, 2000)

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?Kinetic of gas production differed between species and so Kinetic studies using sheep inoculum cannot not be extrapolated to cattle. ?Should develop one inoculum that will minimize requirement surgically modified animal and involve use of mathematical manipulation to shift GP Kinetics produced by one inoculum to that produce by an alternative standard inoculum. ?Using GPT, protozoa has been reported not to survive after 72 hrs of incubation (Rymer and Givens, 2002)

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?In vivo, Endogenous N sources such as protein and urea fluxes into rumen with saliva/through rumen wall, are not easily represented in the GPT ?In vitro methodology is a batch culture system where neither rumen outflow nor absorption can be simulated.

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Makkar et al. (1995) Blümmel et al. (1997) Menke et al. (1979) Blümmel and Orskov (1993) Improvement

Conclusion : 

Conclusion ?Optimal use of GP data for predicting animal response is best achieved when limitations of the system are recognized and solved. ?IVGPT can be used in routine feed evaluation as it is economical and highly reproducible ?If this method is developed with rumen outflow or absorption, correlation with in vivo will be more.

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THANK YOU

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Mould et al. (2005)

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Effect of omitting reducing solution on organic matter degradation (g/kg) Morgan et al. (2004)

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Cumulative gas release (ml/g organic matter incubated) Morgan et al. (2004)

Partitioning Factor : 

Partitioning Factor The ratio of in vitro substrate truly degraded to gas; could serve as an index to assess the differences in EMBS of feedstuffs PF = mg truly degraded OM/ml gas (Blummel et al. 1997) PF is a measure of efficiency of microbial mass production or efficiency of microbial protein production. Higher the PF, higher the efficiency (Makkar, 2001) Ingredient selection for higher PF - higher EMBS. ( Kiran and krishnamoorthy 2007)

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Partitioning of truly degradable substrate between microbial biomass and SCFA+ gases Gas volume derived from 130 mg depends on microbial efficiency. High YATP results in production of more microbial biomass - can be assessed by high PF

In vivo: MCP (g/day)=131.7 ×renal allantoin-N (g/day) Renal Allantoin Excretion (mmol/day ×56)= renal allantoin-N (mg/day) EMBP=g MCP/Kg DOMI Or g MCP/MJ MEI ( Ehrentreich, 1992) : 

In vivo: MCP (g/day)=131.7 ×renal allantoin-N (g/day) Renal Allantoin Excretion (mmol/day ×56)= renal allantoin-N (mg/day) EMBP=g MCP/Kg DOMI Or g MCP/MJ MEI ( Ehrentreich, 1992) Depressed microbial activity when reducing solution was included, its use should be modified. (Fukushima et al., 2003)

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Samples were obtained from fifty-four roughages and roughage neutral-detergent fibres. Y = volume of gas produced (ml), t = time of incubation (h) and A, B, c, ANDF, BNDF &, CNDF are constants. LW-live weight, * Partial R2 Stepwise multiple correlations (R2) between the parameters A,B, (A+B), c, ANDF, BNDF, (ANDF+BNDF) and cNDF as the model Y=A+B(1-e-ct) and the DMI of 54 roughages ( Blümmel et al., 1997)

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Difference (%) between the observed gas production of the mixtures of untreated and (NH4)HCO3- treated rice straws with ryegrass hay and mulberry leaves, and that predicted from feedstuffs fermented seperately

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Chemical composition, cumulative gas production at 24h in vitro incubation with rumen inoculum and predicted energy content from different feedstuff (Krishnamoorthy, et al. 1995) *

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Gas volume = mmol of gas x gas constant (R) x T where: R = the ratio between molar volume of gas to temperature (Kelvin zero; K) i.e. (22.41 l/273 = 0.082) T = incubation temperature (Kelvin); 273 + 39o C = 312 K Total volume of gas (ml) calculated from SCFA production = (BG +FG) x CF BG = gas volume (ml) from buffering of SCFA FG = fermentative gas (ml) (CO2 + CH4) CF = correction factor for altitude and pressure which is 0.953 for Hohenheim at altitude 400m above sea level (Blümmel et al., 1999)

Determination of N Degradability : 

Determination of N Degradability IVGPT as an alternative methods was developed to estimate CP degradation in the rumen (Raab, et al., 1983) When a known amount of starch is added to a protein source incubated in syringe, increasing amount of starch affects gas production as well as NH3-N concentration during fermentation

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Schematic representation of the relationship between protein degradation and protein synthesis in rumen fluid in vitro A- NH3-N content after incubation of feedingstuff alone, C- gas production from feedingstuff alone B- NH3-N content after incubation of feedingstuff + starch, D- gas productions from feedingstuff + starch E, F, G- microbial N expected to be derived from NH3-N, amino acids and peptides, when the feedingstuff is incubated alone H- NH3-N expected to be present at zero gas production.

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Linear regression between NH3-N concentration (mg) and gas production , gives an intercept (b0) representing that amount of NH3-N which would be released when no fermentable carbohydrates were available and no bacterial protein synthesis NH3-N at zero gas production (b0),-NH3-N of blank Total N of feedingstuff incubated IVDN=

Correlations (R2) between in vitro gas volumes from fifty-four roughages (?) and roughage neutral-detergent fibre fractions (?) measured at 4,6,8, 12,24,30, 36,48,54,60,72 and 96 h incubation and DMI : 

Correlations (R2) between in vitro gas volumes from fifty-four roughages (?) and roughage neutral-detergent fibre fractions (?) measured at 4,6,8, 12,24,30, 36,48,54,60,72 and 96 h incubation and DMI *

Determination of Microbial Mass : 

Determination of Microbial Mass Microbial mass (mg) = mg microbial N in syringe × 100 / % N in the microbial fraction Where TSD: True substrate degradability ( Blümmel, 2000 ) N Balance approach: Microbial N in syringe (mg)=N in apparently undegraded residue – NDIN at the end of incubation (Makkar, 2005)

In vitro gas production of tannin-containing leaves in absence of PEG (0 PEG) and presence of PEG (+ PEG) : 

In vitro gas production of tannin-containing leaves in absence of PEG (0 PEG) and presence of PEG (+ PEG) (Getachew et al., 2000) Apple ring acacia (Acacia albida), Beach acacia (Acacia cyanophylla) Red calliandra (Calliandra calothyrsus)