ASC 684 VFA Metabolism I. Factors affecting concentrations of VFA A. VFA production B. VFA absorption C. Interconversions among VFA 1. Eg. Acetate/Butyrate II. Factors affecting proportions of VFA A. Forage:concentrate ratio 1. More complex than just cellulose yields more acetate, etc. Stoichiometric relationships for fermentation of substrates degraded in the rumen for roughage and concentrate diets a. 60% Roughage 40% Roughage Substrate Ac Pr Bu Vi A:P Ac Pr Bu Vi A:P Change in A:P (40%:60%) ----mol produced/mol substrate degraded---- ----mol produced/mol substrate degraded---- Cellulose 1.32 0.17 0.23 0.03 7.76 1.58 0.12 0.06 0.09 13.17 1.70 Hemicellulose 1.13 0.36 0.21 0.05 3.14 1.12 0.51 0.11 0.07 2.20 0.70 Protein 0.40 0.13 0.08 0.33 3.08 0.36 0.16 0.08 0.33 2.25 0.73 Starch 1.19 0.28 0.20 0.06 4.25 0.80 0.60 0.20 0.10 1.33 0.31 Soluble CHO 1.38 0.41 0.10 0.00 3.37 0.90 0.42 0.30 0.04 2.14 0.64 a Adapted from Firkins et al. 2006. Integration of ruminal metabolism in dairy cattle. J. Dairy Sci. 89(E. Suppl.):E31 E51. B. Typical VFA concentrations: 70 to 130 mm (lower w/ forage diets/higher w/ high concentrate diets) C. Intake (increasing intake/passage rate assoc. w/ increase in propionate) D. Degradability (increasing starch degradability assoc. w/ increase in propionate) E. Feeding frequency increased frequency of feeding tends to increase A:P F. Use of chemical additives (Figure and Table from Van Nevel and Demeyer, 1988. Manipulation of rumen fermentation. In: Hobson, P.N. (Ed.) The Rumen Microbial Ecosystem. Elsevier Applied Science. London and New York.
1. Ionophore antibiotics (monensin as example) a) Monensin is a carboxylic polyether antibiotic produced by Streptomyces cinnamonensis. b) Moderate in vitro activity against G(+) bacteria c) Ionophore acts as carrier for monovalent cations (high affinity for Na+) Others, like lasalocid, carry divalent cations, as well. d) Also has anticoccidial properties e) Generally see increased feed efficiency (concomitant w/ decreased intake, esp. on concentrate diets) f) Increases propionate production (up to 30%) and molar proportion of propionate & decreases methane production g) Decreases protein degradability (esp. deamination) related to susceptibility of hyperammonia producers to monensin? III. (1) Include Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum VFA production A. Methods for measuring 1. Non tracer methods a) Zero time in vitro method (1) Measure incremental increases in VFA concentrations at different sampling times in vitro (2) Extrapolate to zero time to determine rate of VFA production per unit volume at time sample was taken. (3) Can multiply rates per unit volume by measure of ruminal volume to determine total ruminal production (4) *Rate using this method about 50% of rate using isotope dilution procedures (general decrease in microbial activity in vitro vs. in vivo) b) Perturbation of steady state (1) Measure change in ruminal concentration with infusion of the VFA (2) Assumes disappearance is proportional to pool size (3) At steady state, then, disappearance (D) will equal some rate constant (k) times the quantity of a VFA present, which is its concentration (C) times ruminal volume (V): D = kcv (4) By definition, at steady state, production (P) will equal disappearance: P = kcv (5) Continuous infusion at a constant rate (I: mmol/h) of a particular VFA will establish new steady state: P + I = U = kc V (where U, C and V are the values at the new steady state). (6) Can combine these equations to ultimately solve for production: P = I/[C V /CV 1]
(7) Assumes that acid infusion doesn t perturb the basal fermentation. c) Portal arterial difference (1) Fick Principle (a) amount of a substance taken up or output from an organ per unit time is equal to the arterial concentration minus venous concentration times the blood flow. (b) Must sample (i) Arterial blood (ii) Venous blood (iii) Determine metabolite concentrations (iv) Measure blood flow (a) Many methods exist (i) Ultrasonic (Doppler) (ii) Microspheres (iii) Thermal dilution (iv) Indicator solutions (e.g. PAH) (2) Measures NET transformation (a) Does NOT account for utilization or production of a metabolite across the organ (b) Thus, doesn t work well for assessing VFA production, per se (ruminal epithelial metabolism of VFA). d) Methane production (1) Total methane production can be measured using (a) indirect calorimetry (i) Measures total (i.e. ruminal and hindgut) methane (b) Isotope dilution techniques (c) Tracer gas (SF 6 ) (2) Multiply methane production by ratio of VFA:methane (total or individual VFA) (a) Ratio determined in vitro or stoichiometrically 2. Tracer methods a) Single pool scheme (1) Assumes steady state conditions (2) Assumes no re entry of label into rumen (3) Recommended tracers include (a) 1 or 2 14 C acetate (b) 2 14 C propionate (c) 1 14 C butyrate or 2 3 3 H butyrate (4) See figure (adapted from France and Siddons, 1993)
(a) Rate of VFA production, k prod (a.k.a. Entry Rate) can be calculated from dose rate (k dose ) and specific activity of ruminal VFA at plateau enrichment (s): k prod = k dose /s (b) This equation is generally derived as follows: at steady state, by definition, k prod = k rem ; likewise, at steady state, k dose = s*k rem ; Thus k prod = k rem = k dose /s. (c) That the removal rate of specific activity equals s*k rem can be seen in the figure if VFA are being removed at a rate of x mmol/h and the enrichment of that VFA pool is y µci/mmol, then the removal rate of the label will be x*y. (5) Gives rate of total VFA production rates for individual VFA can be estimated by measuring proportions of each in the total VFA and assuming that production is proportional to concentration (a) Some have suggested that his assumption works better for forage diets than for concentrate diets. However, recent work by Sutton et al. (2003) J. Dairy Sci. 86:3620 indicates that forage:concentrate ratio may not be important. They found that, for acetate and propionate, concentration proportions gave good proxy of production proportions less so for butyrate.
(6) This method can be modified to use a pulse dose of tracer rather than continuous infusion b) Three pool scheme (1) Not dependent on the proportionality between VFA production and concentration From France and Siddons, 1993. (2) Tracer administeredd to each compartment in turn and with each dose, sa of all compartments is measured.
(3) Series of simultaneous equations is solved to determine movement of tracer and tracee between compartments (4) Results from a study using this approach (Sutton et al., 2003) are shown here
Key points from this graphic: Shift from normal diet (40:60 roughage:concentrate) to low roughage (10:90 roughage:concentrate) diet resulted in large increase in propionate production and removal rates, with relatively small effects on acetate and butyrate. Interconversions with propionate are relatively small, whereas considerable interconversions occur between acetate and butyrate (with both diets, the net consequence of these transactions was a net production of butyrate from acetate). Other data from that study: