6 Volatile Fatty Acid Production J France1 and J Dijkstra2 Centre for Nutrition Modelling, Department of Animal & Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada; 2Animal Nutrition Group, Wageningen Institute of Animal Sciences, Wageningen University, PO Box 338, 6700 AH Wageningen, The Netherlands Introduction Volatile fatty acids (VFAs), principally acetate, propionate and butyrate but also lesser amounts of valerate, caproate, isobutyrate, isovalerate, 2-methylbutyrate and traces of various higher acids, are produced in the rumen as end-products of microbial fermentation During the fermentation process energy is conserved in the form of adenosine triphosphate and subsequently utilized for the maintenance and growth of the microbial population As far as the microbes are concerned the VFAs are waste products but to the host animal they represent the major source of absorbed energy and with most diets account for approximately 80% of the energy disappearing in the rumen (the remainder being lost as heat and methane) and for 50–70% of the digestible energy intake in sheep and cows at approximately maintenance, the range being 40–65% in lactating cows (Sutton, 1972, 1979, 1985; Thomas and Clapperton, 1972) Dietary carbohydrates, i.e cellulose, hemicellulose, pectin, starch and soluble sugars, are the main fermentation substrates They are degraded to their constituent hexoses and pentoses before being fermented to VFA via pyruvate (Fig 6.1) Pentoses are converted to hexose and triose phosphate by the transketolase and transaldolase reactions of the pentose cycle so that the majority of dietary carbohydrate metabolism proceeds via hexose, which is metabolized to pyruvate almost exclusively by the Embden–Meyerhof glycolytic pathway Acetyl CoA is an intermediate in the formation of both acetate and butyrate from pyruvate, whilst propionate formation occurs mainly via succinate although an alternative pathway involving acrylate is also operative The need to maintain redox balance through reduction and reoxidation of pyridine nucleotides (NAD) controls fermentation reactions (review Dijkstra, 1994) Excess reducing power generated during the conversion of hexose to acetate or butyrate is utilized in part during the formation of propionate but mainly by conversion to methane The overall reactions can be summarized as: ß CAB Internatioal 2005 Quantitative Aspects of Ruminant Digestion and Metabolism, 2nd edition (eds J Dijkstra, J.M Forbes and J France) 157 158 J France and J Dijkstra Hemicellulose Pectin Cellulose Starch Soluble sugars Pentoses Pentose cycle Hexoses Embden−Meyerhoff pathway Pyruvate Formate Acrylate pathway Acetyl CoA Succinate pathway CO2 + H2 Methane Fig 6.1 rumen Acetate Butyrate Propionate A schematic representation of the major pathways of carbohydrate metabolism in the hexose ! pyruvate þ 4H pyruvate þ H2 O ! acetate þ CO2 þ 2H pyruvate ! butyrate þ2CO2 pyruvate þ 4H ! propionate ỵ H2 O CO2 ỵ 8H ! methane þ 2H2 O In addition to dietary carbohydrates, dietary lipids and proteins also give rise to VFAs in the rumen The contribution from lipids is very small as lipids normally represent a small proportion of the diet and only the carbohydrate moiety, i.e glycerol and galactose arising from lipid hydrolysis, and not the longchain fatty acids, are fermented Dietary proteins on the other hand may be a significant source of VFA when diets having a high rumen-degradable-protein content are fed The proteins are hydrolysed to amino acids, which are deaminated before conversion to VFA Of particular importance in this respect is the formation of isobutyric, isovaleric and 2-methylbutyric acids from valine, leucine and isoleucine, respectively, as these branched-chain VFAs are essential growth factors for certain of the rumen bacterial species (Cotta and Hespell, 1986) The majority of the VFAs produced in the rumen are lost by absorption across the rumen wall, although a proportion (10–20% in sheep and up to 35% in dairy cattle) pass to the omasum and abomasum and are absorbed from these organs (Weston and Hogan, 1968; Dijkstra et al., 1993) Absorption across the rumen wall is by simple diffusion of the undissociated acids (Stevens, 1970; Dijkstra et al., 1993) It is a concentration-dependent process and therefore Volatile Fatty Acid Production 159 (of the three major VFAs) usually higher for acetate than for propionate and lowest for butyrate, but per unit of concentration the absorption rates of the three acids are quite similar, although at low pH VFA with a higher carbon chain have a higher fractional absorption rate due to their greater lipid solubility (Dijkstra et al., 1993; Lopez et al., 2003) As the pKa values of the acids are lower than the pH of rumen contents, they exist largely in the anionic form A fall in rumen pH is associated with an increase in the proportion in the undissociated form and therefore in the rate of absorption During passage across the rumen wall the VFAs are metabolized to varying extents so that the amounts entering the bloodstream are less than the quantities absorbed from the rumen (Weigland et al., 1972; Bergman, 1975; Weekes and Webster, 1975) However, recent results in which VFA absorption from the temporarily isolated and washed rumen was compared with the portal VFA absorption indicate that the rumen wall does not metabolize large amounts of acetate, propionate and isobutyrate absorbed from the rumen, though the extensive metabolism of butyric acid during absorption was confirmed (Kristensen et al., 2000) The concentration of VFA in the rumen at any given time reflects the balance between the rate of production and rate of loss Immediately after feeding, production exceeds loss and the concentration increases, but subsequently the situation is reversed and the concentration falls The total VFA concentration may fall as low as 30 mM or be in excess of 200 mM but is normally between 70 and 130 mM The relative concentrations of the individual acids, commonly referred to as the fermentation pattern, is a reliable index of the relative production rates of the acids when forage diets are given but would appear less reliable with concentrate diets (Leng and Brett, 1966; Esdale et al., 1968; Sharp et al., 1982; Sutton, 1985) The fermentation pattern is determined by the composition of the microbial population, which in turn is largely determined by the basal diet, particularly the type of dietary carbohydrate, and by the rate of depolymerization of available substrate (review by Dijkstra, 1994) High-fibre forage diets encourage the growth of acetateproducing bacterial species and the acetate:propionate:butyrate molar proportions would typically be in the region 70:20:10, whereas starch-rich concentrate diets favour the development of propionate-producing bacterial species and are associated with an increase in the proportion of propionate at the expense of acetate, although acetate is almost always the most abundant of the acids Under certain conditions, concentrate diets may encourage the development of a large protozoal population and this is accompanied by an increase in butyrate rather than propionate (Williams and Coleman, 1997) If levels of substrate available for fermentation are high, either from increased intake or increased rates of depolymerization, a shift in fermentation pattern from acetic acid to propionic acid occurs to dispose of excess reducing power (Dijkstra, 1994) In addition to the type of dietary carbohydrate, other factors such as the physical form of the diet, level of intake, frequency of feeding and the use of chemical additives may also affect the fermentation pattern (Ørskov, 1981; Thomas and Rook, 1981; Nagaraja et al., 1997) Some examples of the fermentation pattern, VFA concentration and production rate in animals 160 J France and J Dijkstra receiving different diets are shown in Table 6.1 More detailed reviews of the various aspects of VFA production and metabolism are given by Bergman (1990) and Dijkstra (1994) Within the host animal’s tissues absorbed acetate and butyrate are used primarily as energy sources through oxidation via the citric acid cycle Acetate is also the principal substrate for lipogenesis, whilst propionate is used largely for gluconeogenesis and with most diets is the major source of glucose, since net absorption of glucose from the intestinal tract is usually small The balance between the supply of the glucogenic propionate relative to that of the non-glucogenic acetate and butyrate influences the efficiency with which the VFAs are used for productive purposes (Ørskov, 1975; MacRae and Lobley, 1982; Sutton, 1985) Thus, not only the total supply of VFA but also the molar proportions are important determinants of feed utilization by ruminants and as such a number of methods have been used to estimate the rates of individual and total VFA production in and removal from the rumen These may be conveniently divided into two groups: Those methods not employing isotopic tracers (e.g Barcroft et al., 1944; Hungate et al., 1960; Bath et al., 1962) Those employing tracers and based on the application of compartmental analysis to interpret isotope dilution data (e.g Bergman et al., 1965; Weller et al., 1967; Morant et al., 1978; Armentano and Young, 1983) Non-tracer Methods of VFA Production Measurement A variety of non-tracer methods of measurement were used in early attempts to quantify VFA production in the rumen, and these are comprehensively reviewed by Warner (1964) and Hungate (1966) They include: the zero-time in vitro method, perturbation of the steady state, portal–arterial difference and methane production Due to interconversions between individual VFA, particularly between acetate and butyrate, the net production rates of the acids (i.e the amounts lost by absorption and passage) are less than the total production rates (Bergman et al., 1965) In this and subsequent sections of the chapter, the term production is synonymous with net production unless total production is specified Zero-time in vitro method A sample of rumen contents is taken and subsamples incubated in vitro under anaerobic conditions The rate of production of individual or total VFAs is calculated from the increments in acid concentration obtained by incubating the subsamples for different periods and extrapolating back to zero time to give the rate of VFA production per unit volume at the time the sample was removed Equations for performing the calculation are given by Whitelaw et al (1970) If the rumen volume is known, total ruminal production can Animal species Sheep Steers Intake (kg/day) Total VFA concentration (mmol/l) Acetate (molar %) Propionate (molar %) Butyrate (molar %) VFA production (mol/day) Dried grass Dried grass 0.89a 0.73b 106 87 68 68 19 21 13 11 5.8 4.08 Dried forage oats 0.78b 100 68 21 11 4.90 Dried clovers 0.97b 118 71 19 10 6.32 Lucerne silage Lucerne chaff Maize:lucerne chaff (2:1) Maize:lucerne chaff (1:1) Lucerne hay:concentrate (4:1) Lucerne hay:lucerne pellets:concentrate (1:3:1) Concentrate:lucerne hay (4:1) Concentrate:lucerne hay:lucerne pellets (16:1:3) Maize silage:concentrate (1:1) Concentrate:maize silage (3:1) Lucerne hay:maize silage:concentrate (3.6:1:1) 0.87c 0.8c 0.6c 0.6c 7.99a 85 131 113 73 103 72 73 63 65 73 22 18 24 21 18 13 14 4.50 4.97 3.61 3.11 50.1 8.29a 100 72 18 10 42.4 8.56a 108 67 22 12 54.1 8.94a 118 63 26 12 42.3 5.19a 123 55 34 11 14.3 7.7a 125 57 31 12 48.3 9.0a 92 72 17 11 33.3 Diet Reference 161 Bergman et al (1965) Weston and Hogan (1968) Weston and Hogan (1968) Weston and Hogan (1971) Siddons et al (1984) Leng and Brett (1966) Leng and Brett (1966) Leng and Brett (1966) Siciliano-Jones and Murphy (1989) Siciliano-Jones and Murphy (1989) Siciliano-Jones and Murphy (1989) Siciliano-Jones and Murphy (1989) Rogers and Davis (1982a) Rogers and Davis (1982b) Rogers and Davis (1982b) continued Volatile Fatty Acid Production Table 6.1 VFA concentration, molar proportions and production rates in the rumen of sheep, steers and cows given various diets 162 Table 6.1 Animal species Dairy cows continued Diet Whole maize:other (5.25:1) Ground maize:other (5.25:1) Lucerne hay:grain (1:1.3) Lucerne hay:grain (1:6.6) Maize silage Lucerne hay Ryegrass hay:concentrate (6:4) Ryegrass hay:concentrate (1:9) Total VFA concentration (mmol/l) Acetate (molar %) Propionate (molar %) Butyrate (molar %) VFA production (mol/day) 6.22 6.22a 19.1c 17.27c 3.5a 3.9a 12.9a 145 141 109 121 83 77 85 49 41 67 49 64 73 68 34 49 21 40 19 17 19 17 10 12 11 17 10 13 51.4 42.0 37.52 44.58 30.9 26.7 79.8 Sharp et al (1982) Sharp et al (1982) Davis (1967) Davis (1967) Esdale et al (1968) Esdale et al (1968) Sutton et al (2003) 12.7a 89 52 38 90.0 Sutton et al (2003) Intake (kg/day) a Reference a Dry matter Organic matter c Not specified b J France and J Dijkstra Volatile Fatty Acid Production 163 then be calculated As with other in vitro techniques, it is important that the sample taken for incubation is representative of whole-rumen contents rather than just the solid or liquid fraction (Hungate et al., 1960) However, the VFA concentrations and molar proportions in in vitro systems often not resemble those in vivo (Mansfield et al., 1995; Ziemer et al., 2000) Whitelaw et al (1970), in comparing published experiments, show that the rate of VFA production determined by this method is about 50% lower than the rate obtained using isotope dilution procedures They attribute the discrepancy to a reduction in the activity of microorganisms brought about by their removal from the rumen Perturbation of the steady state The rate of total production of an acid (or net production of total VFA) in the rumen in steady state can be calculated from the change in its ruminal concentration when the acid is infused Let P (mmol/h) be its rate of production, U (mmol/h) its rate of disappearance and C (mmol/ml) its concentration in the basal steady state Assuming disappearance is proportional to acid pool size, the balance equation may be written as: P ¼ U ¼ kCV (6:1) where k (per h) is a constant of proportionality and V (ml) the ruminal volume Let the basal steady state be perturbed by infusion of a solution of the acid at a constant rate I (mmol/h) such that a new steady state is reached If the acid infusion does not alter the basal fermentation, the balance equation in the new steady state is: P ỵ I ẳ U ¼ kC0 V (6:2) where U , C0 and V denote acid utilization, acid concentration and ruminal volume, respectively, in the new steady state Subtraction of Eq (6.1) from Eq (6.2) yields an expression for the constant of proportionality: k ¼ I=(C0 V
CV) (6:3) Substituting for k in Eq (6.1) gives the rate of production: P ¼ I=[C0 V =(CV)
1] (6:4) The steady-state volumes V and V can be determined using one of the methods, based on digesta markers and intraruminal sampling, described in France et al (1991a) This approach of raising the steady-state level was used by Bath et al (1962) though they assumed a constant ruminal volume and expressed the acid concentration relative to that of the other acids Martin et al (2001) adopted the perturbation of steady-state method with some modifications They infused VFA 164 J France and J Dijkstra into the rumen at five levels and estimated VFA production using a regression approach They observed that the VFA production rate obtained with the regression approach was about two-thirds of that obtained with the isotope dilution technique This difference may be explained to an extent by the use of 1-13 C propionate because of the labile nature of the carboxyl-C A critical assumption in the perturbation of steady-state method is that the rate parameter k is not altered by the acid infusion However, a change in VFA concentration and other modifications that result from the acid infusion, including a change in pH, affect the fractional absorption rate of VFA (Dijkstra et al., 1993) and consequently k values may differ Portal–arterial difference in VFA concentration The difference between VFA concentration in venous blood draining the rumen and that in arterial blood provides a measure of the amount entering the blood from the rumen, if the rate of blood flow is known Vessels normally sampled are the portal vein and the carotid artery This method was used by Barcroft et al (1944) to demonstrate that acids from the rumen fermentation are absorbed and utilized by the host Metabolism of VFA in the rumen wall, however, precludes accurate estimation of ruminal VFA production Bergman (1975) estimated that in sheep receiving a forage diet, approximately 90% of the butyrate, 50% of the propionate and 30% of the acetate produced in the rumen did not appear in the portal blood These values were generally in good agreement with in vitro data on the loss of VFA transported across the rumen epithelium (review Re´mond et al., 1995) However, Kristensen et al (2000) observed considerably higher recovery rates of acetate and propionate in the temporarily isolated rumen of sheep To explain the differences, Kristensen et al (2000) suggested substantial microbial utilization of VFA Also, measurements of blood flow show considerable variability (Dobson, 1984) Methane production Methane production is an index of rumen fermentation, which has been used to obtain indirect estimates of VFA production Total methane production can be measured in intact, non-fistulated animals using indirect calorimetry (McLean and Tobin, 1987) or the polytunnel method (Lockyer and Jarvis, 1995) Calorimetry and the polytunnel, however, overestimate the ruminal contribution; Murray et al (1976), for example, showed that the production of methane in the rumen of sheep fed lucerne chaff accounted for 87% of the total production Alternatively, ruminal methane production can be measured with fistulated animals using isotope dilution techniques (Murray et al., 1976, 1978; France et al., 1993) Also, non-isotopic tracer techniques have been developed to measure ruminal methane production in free-moving, intact animals, such as the sulphur hexafluoride (SF6 ) method (Johnson et al., 1994) The value obtained for methane production is then multiplied by the Volatile Fatty Acid Production 165 ratio of individual or total VFA produced to methane produced This ratio may either be determined in vitro using rumen samples, or calculated stoichiometrically (Murray et al., 1978), provided the VFA proportions are known The method relies on a close relationship between VFA and methane produced, based on the need to maintain redox balance in the rumen However, a number of other factors, including the uptake of hydrogen for biohydrogenation of unsaturated long-chain fatty acids and the uptake or release of hydrogen for microbial protein synthesis, may impair this relationship (Mills et al., 2001) Tracer Methods of VFA Production Measurement The tracer methods developed in this section are described for radioactive isotopes, though they are equally valid for stable isotopes (see end of section, page 171) For measurement of VFA production by radioactive isotopic tracer techniques, Bruce et al (1987) recommended the use of or 2-14 C acetate, 2-14 C propionate and 1-14 C butyrate 2-33 H butyrate may also be used (Leng and Brett, 1966), but 2-3 H acetate is unsatisfactory (Leng and Leonard, 1965) Single-pool scheme A relatively simple approach, which assumes steady-state conditions as imposed by continuous feeding, was proposed by Weller et al (1967), whereby total VFA is considered to behave as a homogeneous pool and therefore can be represented as a single-pool model (Fig 6.2) The isotopic form of any one of the individual VFAs or a mixture of the VFAs is administered into the rumen by continuous infusion at a constant rate, I (mCi=h), and the plateau specific activity of the total VFA, s (mCi=mmol), is subsequently determined from the isotope concentration (mCi=ml) and total VFA concentration (mmol/ml) in rumen liquid The rate:state equations, based on mass conservation principles, for this steady-state scheme are: dQ ¼ Fvo
Fov dt dq ¼ I
sFov dt (a) (6:5) (6:6) (b) Fvo VFA, Q Fov I q sFov Fig 6.2 Single-compartment model for estimating VFA production: (a) tracee and (b) tracer The scheme assumes no re-entry of label into the rumen Q, total VFA; q, quantity of tracer; Fvo , rate of de novo VFA production; Fov , rate of VFA removal; s, plateau specific activity of total VFA; and I, infusion rate 166 J France and J Dijkstra where Q (mmol) denotes total VFA, q (mCi) the quantity of tracer, Fvo (mmol/h) the rate of production de novo (i.e entry into the pool) and Fov (mmol/h) the rate of removal The g carbon can equally well be used instead of the mmol as the unit of mass On solving Eqs (6.5) and (6.6), the rate of VFA production becomes: Fvo ¼ I=s (6:7) The production rate of the individual VFA is then obtained from their respective concentrations in the rumen liquid by assuming that production is proportional to concentration, e.g Rate of acetate production ¼ Fvo Ca =Cv (6:8) where Ca and Cv (both mmol/ml) are the concentrations of acetate and total VFA, respectively Assuming isotope concentration and total VFA concentrations are measured in a number of samples, then the rate of VFA production may be calculated from Eq (6.7) using either the mean specific activity or the specific activity of a pooled sample or, alternatively, by multiplying the infusion rate by the mean reciprocal specific activity Although with steady-state conditions all three procedures should give the same result, Morant et al (1978) found in simulation studies with non-steady-state conditions that estimates obtained using the latter procedure were closer to the true production rates and recommended its use in preference to P the other two (Note: Eq (4) in Morant et al (1978) should read MR ¼ (IR =n) ni¼1 Mi =Ii :) Weller’s method can be adapted for single-dose injection of tracer, rather than continuous infusion Equation (6.6) reduces to: dq ¼
sFov dt (6:9) where s is now the instantaneous specific activity Integration of Eq (6.9) with respect to time between time zero and infinity gives:
D ¼
AFov (6:10)  Ð1  where D (mCi) is the dose injected at time zero and A ¼ sdt denotes the area under the VFA specific activity–time curve As the rate of removal equals that of production in steady state, then: Fvo ¼ D=A (6:11) i.e the rate of VFA production equals dose over area under the specific activity–time curve When the system is not in steady state (i.e with animals that are not continuously fed), the VFA pool size, Q, and the production rate will vary ... 7.99a 85 131 113 73 103 72 73 63 65 73 22 18 24 21 18 13 14 4 .50 4.97 3.61 3.11 50 .1 8.29a 100 72 18 10 42.4 8 .56 a 108 67 22 12 54 .1 8.94a 118 63 26 12 42.3 5. 19a 123 55 34 11 14.3 7.7a 1 25 57 31... R.R and Owens, F.N (1982) Ruminal VFA production with steers fed whole or ground corn grain Journal of Animal Science 55 , 150 5– 151 4 Siciliano-Jones, J and Murphy, M.R (1989) Production of volatile... Murphy (1989) Siciliano-Jones and Murphy (1989) Siciliano-Jones and Murphy (1989) Siciliano-Jones and Murphy (1989) Rogers and Davis (1982a) Rogers and Davis (1982b) Rogers and Davis (1982b) continued