Ruminal Ingestat. Madison, Wisconsin and amount of end-product formation from. as donors of ruminal ingesta. The first cow received

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1 APPI WF MICROBIOLOGY, May 1968, p Vol. 16, No. 5 Copyright 1968 American Society for Microbiology Printed in U.S.A. In Vitro Lactate Metabolism by Ruminal Ingestat L. D. SAlTER AND W. J. ESDALE Dairy Science Department, University of Wisconsin, Madison, Wisconsin 5376 Received for publication 11 December 1967 Ruminal ingesta (3 ml) obtained from a fistulated cow fed alfalfa hay (H), 3.6 kg of grain mixture with corn silage fed ad libitum (S), 2.5:1 grain-alfalfa hay mixture (G), or a 2.5:1 grain-alfalfa hay mixture providing 545 g of sodium and calcium lactate daily (L) were incubated for 8 hr with nonpolymerized sodium lactate or 17% polymerized lactic acid neutralized toph 6.7. Polymerization had no effect on the rate of lactate utilization. The initial rates of lactate metabolism for the H, G, S, and L ingesta were.72,.95, 1.8, and 3.4 meq per 1 ml of rumen fluid per hr, respectively. Lactate-2-'4C was incubated for 4 hr with each type of ruminal ingesta. Of the label recovered in the volatile fatty acids (VFA), 74.1, 61.2, 49.3, and 38.9% was recovered in acetate, and 9.4, 19.8, 23.3, and 5% was recovered in propionate with H, G, S, and L ingesta, respectively. The balance of label was distributed between butyrate and valerate. The titratable VFA did not follow this pattern of production. With the hay ingesta, lactate metabolism resulted in a net loss of acetate and a large increase in butyrate. Little propionate was produced. The G, S, and L ingesta metabolized lactate to yield progressively more propionate and less butyrate. Evidence was gathered to suggest that acetate was the primary end product of lactate metabolism but that oxidation of lactate to pyruvate dictated the synthesis of butyrate from acetate to maintain an oxidation-reduction balance. It was noted that acetate and butyrate production from lactate was ph-dependent, with acetate production maximal at ph 7.4 and butyrate at 6.2. Propionate production was largely unaffected within this ph range. Lactic acid frequently accounts for 3 to 8% of the dry matter of corn or grass silage. This would result in an intake of.5 to 1 lb (.27 to.453 kg) of lactic acid daily per animal in many dairy and beef cattle rations. Previous investigations of lactate metabolism by the rumen microflora have led to contradictory conclusions. Several studies have suggested that the principal end product of lactate fermentation by the rumen microflora is propionate. These observations have resulted in attempts to feed lactate salts as a therapeutic agent in the treatment of ketosis in dairy cattle. Presumably, increased ruminal propionate production would enhance glucogenesis in the animal cell, thus mitigating the ketotic state. The use of lactate salts has been highly unsuccessful in treating ketosis, probably because most studies suggest that acetate or butyrate may be the principal end product of lactate metabolism by rumen microorganisms. 1 Pubhshed with the approval of the Director of the Wisconsin Agricultural Experiment Station. Our inadequate understanding of lactate metabolism by the rumen microflora, in spite of the prominent role lactate plays as a silage ingredient and as an intermediate in the formation of some ruminal volatile fatty acids (VFA), suggested further examination of lactate metabolism. All experiments reported herein were carried out in vitro. They were designed to characterize the rate and amount of end-product formation from lactate when incubated with ruminal ingesta obtained from cows receiving four different rations. Downloaded from on April 2, 218 by guest MATERIALS AND METHODS Experiment 1. Four cows fitted with rumen cannulae, each of which received a different ration, served as donors of ruminal ingesta. The first cow received second-cutting alfalfa hay fed ad libitum, and the second cow received corn silage fed ad libitum (32 to 4 kg daily) plus 3.6 kg of concentrate mixture (shelled corn, kg; oats, kg; soybean oil meal, 45.5 kg; dicalcium phosphate, 4.5 kg; and tracemineralized salt, 4.5 kg). The third cow received

2 VOL. 16, 1968 LACTATE METABOLISM'. BY RUMINAL INGESTA 681 parts concentrate and 1 part alfalfa hay fed ad libitum, and the fourth cow received 3.9 kg of concentrate mixture plus 2.3 kg of alfalfa hay and 1.8 kg of Ketolac (a product of Foremost Dairies, formerly Western Condensing Co., Appleton, Wis.; it contained 3 to 35% sodium and calcium lactate salts, 2% wheat bran, and 45 to 5% whey). The cows were fed at 7:3 AM and 3:45 PM. Ruminal ingesta containing a considerable quantity of solid feed particles were obtained from both the ventral and dorsal regions of the rumen between 11: and 11:3 AM. Unstrained ruminal ingesta (3 ml) were mixed with 34.5 meq of either 17% polymerized lactic acid neutralized to ph 6.7 or nonpolymerized sodium lactate. Degree of polymerization was determined by titration before and after refluxing with N NaOH under nitrogen for 3 min. The incubation flasks were 4-ml jars fitted with rubber stoppers containing Bunsen valves. After addition of the lactate and ingesta to the jars, a 7:3 mixture of C2-methane was used to flush residual air prior to inserting stoppers in the flasks. The vessels were incubated for 8 hr at 37 C and shaken gently every 2 hr. Incubation vessels containing polymerized or nonpolymerized lactate were compared to control vessels containing no lactate. The difference in measured VFA production between the control and experimental flasks was attributed to the added lactate. Treatment and control incubations were run in duplicate on 3 different days, with the exception of the silage ingesta where two donor cows were used and a total of 1 incubations were run. Microbial activity was stopped at the end of 8 hr by acidification to ph 2 with H2SO4. After filtration of the incubated ingesta through two layers of cheesecloth and centrifugation at 17, X g for 15 min, 15-ml samples of the supernatant liquid were steamdistilled to measure total VFA. The relative amounts of VFA were determined by gas chromatography (5), and the procedure of Barker and Summerson (4) was used for lactate analysis. Experiment 2. Radioisotope studies were conducted to examine the distribution of lactate carbon between the VFA and to note the effect of lactate on the randomization ot carbon between each of the VFA. In the first study,.69 meq of sodium lactate-2-14c was incubated for 4 hr with 5 ml of each of the four types of ruminal ingesta described earlier. The shorter incubation time of 4 hr was used to minimize randomization of carbon between the VFA. The sampling and incubation conditions were similar to those described in experiment 1. After acidification to halt the fermentation, the ingesta were adjusted to ph 8 and transferred to a Buchner funnel lined with a 1- mesh nylon screen, filtered, and washed successively with.1 N NaOH and HCI. The washed residue and nylon screen were dried and combusted in a Thomas- Ogg combustion flask (Arthur H. Thomas Co., Philadelphia, Pa.), and the collected CO2 was counted in a liquid scintillation spectrometer. The filtrate was centrifuged at 17, X g for 15 min. The pellet obtained upon centrifugation was washed three times with.9% saline; the last two washings con- tained.5 N NaOH and HCI, respectively. The remaining residue, containing most of the bacteria and protozoa, was dried and combusted in a Thomas-Ogg combustion flask. The supernatant fluids and washings were combined, acidified, and steam-distilled. A sample of the nonvolatile fraction was prepared for counting in the liquid scintillation spectrometer. The distillate was adjusted to a basic ph and reduced in volume to 5 ml by evaporation. A 2-ml sample of this fraction was added to a Wiseman and Irwin (29) liquid-liquid column chromatograph, and each of the VFA fractions was collected, titrated, and counted in a liquid scintillation spectrometer. The results represent an average of two incubations done on different days for the hay and silage ingesta, whereas only one incubation was made for the grain and lactate ingesta. In the second study, the effect of lactate on the randomization of VFA carbon was examined by incubating tracer amounts of acetate-2-14c, propionate- 3-14C, or butyrate-1-14c with 25 ml of hay, grain, silage, or lactate ingesta in the presence or absence of 1.3 meq of sodium lactate. The incubation lasted 8 hr, but a subsample was obtained after 4 hr from each of the incubation vessels. After acidification to halt fermentation, the ingesta was filtered through two layers of cheesecloth and centrifuged at 17, X g for 15 min. The VFA in the supernatant liquid were separated on a Wiseman and Irwin (29) column, and the effluents were titrated and counted to determine the extent of carbon randomization between each of the VFA. Experiment 3. The effect of the presence or absence of acetate, propionate, or butyrate on lactate metabolism by ruminal ingesta was investigated. Ruminal ingesta, collected 2 hr after feeding from a cow maintained on an alfalfa hay ration, were filtered through cheesecloth, acidified, and steam-distilled to remove the VFA. The distillate was discarded, and the nonvolatile fraction was adjusted to ph 8. and agitated with CO2 until the ph was 6.7. etate, propionate, or butyrate was individually added back to the nonvolatile rumen liquor to make a 6-mM concentration. An amount of 35 ml of the VFA-enriched rumen liquor was added to a centrifuge tube containing a washed preparation of rumen microbal cells with or without sodium lactate. The final concentration of sodium lactate was 72 mm. The centrifuge tubes were fitted with Bunsen valves and flushed with the C2- CH4 mixture prior to 7 hr of incubation. Two cell preparations were used. Both were prepared from ruminal ingesta taken from the same cow used above, but sampled at 6 hr after feeding. The first was prepared by filtering 4 ml of ruminal ingesta through cheesecloth and centrifuging the filtrate at 17, X g for 5 min. The cells and accompanying feed particles were resuspended and washed once with water before a second centrifugation at 17, X g. Incubations were run in duplicate on 6 different days. The second, cleaner preparation of cells was obtained by centrifuging the filtered ingesta at 6 X g for 5 min to remove many of the feed particles. This undoubtedly removed most of the protozoa and some clumps of bacteria in addition to feed particles. The supernatant liquid was recentrifuged at 17, X g for 5 min and was subjected to two washings with Downloaded from on April 2, 218 by guest

3 682 SA'TER AND ESDALE APPL. MICROBIOL. Li 12 DISAPPEARANCE INGE STA LACTATE DISAPPEARANCE G4 GRAIN INGESTA j 1 o Os 6 ' \S z o a2 -NONPOLYMERIZED W Wo POLYMI2 -EPOL J a --- NONPC o ~~4 S 4 HOURS INCUBATED HOURS INCUBATED LACTATE DISAPPEARANCE LACTATE DISAPPEARI 14,SILAGE INGESTA LACTATE INGE! STA 12' 12 %~~~~~~~ %~~~~~~~ 1z \ to 2IZ z 8 8 S o %~ ~ ~ ~~ Z~~~I6 %~~~~~~~~ ~~~~~~~~ ~24, 4 -POLYIIERIZED %% c~~3- --NONPO)LYMERIZED 2 N-ONPOLYMERZED HOURS INCUBATED HOURS INCUBATED FIG. 1. Rate oflactate metabolism by ruminal ingesta. Downloaded from on April 2, 218 by guest

4 VOL. 16, 1968 LACIATE METABOLISM BY RUMINAL INGESTA 683.9%, NaCl. This preparation was incubated in the same fashion as the other, but it was allowed to go 24 hr rather than 7 hr. Incubations were run in duplicate on 2 different days. Experiment 4. VFA production from sodium lactate and sodium pyruvate was determined by incubating 3.4 meq of each salt for 8 hr with 5-ml samples of hay ingesta. As in experiment 1, the difference in VFA production between the two treatments and a control was attributed to the added lactate or pyruvate. Treatment and control incubations were run in duplicate on 2 different days. VFA analysis and incubation conditions were the same as described in experiment 1. Experiment 5. The effect of ruminal ingesta ph on VFA production in the presence of lactate was examined. A 5-ml amount of unstrained hay or grain ingesta was incubated for 4 hr with 3.4 meq of sodium lactate at ph 8., 7.4, 6.8, 6.2, 5.6, or 5.. The ph designated was maintained by frequent additions of dilute solutions of either HCI or NaOH. VFA production was taken to be the difference in VFA concentration between and 4 hr. VFA analysis and incubation conditions were the same as described in experiment l, with the exception that frequent entry into the incubation vessel was necessary to maintain the appropriate ph. RFSULTS The rate of lactate metabolism by the four types of ruminal ingesta is presented in Fig. 1. In all cases, the 17%o polymerized lactate was degraded as rapidly as the nonpolymerized lactate. There was a marked difference between the rate at which the types of ingesta degraded lactate. Lactate ingesta showed the greatest activity, followed in descending order by silage, grain, and hay ingesta. Since there appeared to be no difference in the rate at which the polymerized and nonpolymerized lactates were degraded, the values for VFA production from the two were combined (Table 1). As indicated by the negative value obtained for acetate with the hay and silage ingesta, a net decrease in acetate production was observed over the 8-hr incubation period. After incubation of lactate-2-14c with each of the four types of ingesta for 4 hr, 72 to 93% of added lactate-2-14c activity was recovered in the VFA fractions, whereas 2 to 13%C was recovered in the nonvolatile fraction. Added activity (3 to 6%) was recovered in the microbial cell fraction, and about the same amount was found adhering to the feed particles. Table 2 gives the distribution of radioactivity recovered in the VFA fraction. In three types of ingesta, acetate contained the majority of label. However, when lactate ingesta were used, propionate contained over 5% of the radioactivity. The percentage of label appearing in acetate was highest for the hay ingesta and decreased in descending order with the grain, silage, and lactate ingesta. The percentage of TABLE 1. id VFA production from added lactate during 8-hr incubation a Ingesta Hay Silage Grain Lactate etate Propionate Butyrate Isovalerate Valerate a Represents the difference (expressed in milliequivalents per 1 ml) in VFA production between control flasks containing no lactate and experimental flasks containing lactate. TABLE 2. Percentage distribution of lactate-2-'4c carbon recovered in the VFA fraction after 4-hr incubation id Ingesta Hay Silage Grain Lactate etate Propionate Butyrate Valerate label appearing in propionatewas inversely related to that of acetate. The extent of randomization of carbon among the VFA was affected to a large degree by the presence of lactate. Lactate greatly enhanced the incorporation of extracellular acetate into butyrate, particularly with hay ingesta (Table 3). Lactate impaired the reverse of this reaction, reducing the extent of extracellular butyrate conversion to acetate. Randomization of propionate carbon was not affected by lactate. The presence of extracellular acetate enhanced the metabolism of lactate. Maximal lactate utilization occurred when acetate was added back to the medium (Table 4). Very little lactate was metabolized when no extracellular acetate was present. The addition of butyrate very likely resulted in the formation of some acetate, so the extent of lactate degradation was not impaired to the same degree with butyrate as it was with propionate. As in earlier experiments, lactate addition led to propionate and butyrate production, with the relative amounts of each varying with the type of ingesta used (Table 5). Pyruvate, in contrast to lactate, yielded essentially acetate with all three types of ingesta employed. The effect of ph on VFA production from lactate is presented in Table 6. The values shown represent total VFA production, not only that Downloaded from on April 2, 218 by guest

5 684 SAlTER AND ESDALE TABLE 3. Randomization of VFA carbona APPL. MICROBIOL. Radioactive compounds Time Hay ingesta Grain ingesta Lactate ingesta With lactate Without lactate With lactate Without lactate With lactate Without lactate etate-2-'4c Propionate-3-14 C Butyrate-1-'4C Bu Pr Bu Pr Bu Pr Bu Pr Bu Pr Bu Pr a Percentage distribution of activity recovered in the butyrate, propionate. and acetate fractions. Bu represents butyrate; Pr, propionate;, acetate. Lactate had no effect on the incorporation of activity into valerate. After 8-hr incubation, valerate seldom had more than 4% of total VFA activity. TABLE 4. hr Lactate metabolism in presence ofacetate, propionate, or butyrate a Compound added Minimal Washing of cells Extensive etate Propionate Butyrate a Milliequivalents of lactate metabolized per 1 ml of ruminal fluid per 7 hr. from lactate. Optimal ph for total VFA production was between ph 6.2 and 7.4. The optimal ph for acetate production of both hay and grain ingesta was 7.4, for propionate, 6.8, and for butyrate, 6.2. etate and butyrate production were quite sensitive to ph, whereas propionate production was sustained over a broader ph spectrum. Marked reductions in total VFA production occurred at ph 5.6 or lower. DIscussIoN Emery et al. (15) reported that 43% polymerized lactate was metabolized by ruminal ingesta at 5% of the rate expected on the basis of its monomer content. These workers also observed that the rate of L(+)-lactate utilization was linear with concentration. The fact that no differences were observed between the rates of metabolism of polymerized and nonpolymerized lactate in this study may be due to two factors: the degree of polymerization was considerably less in this study, and the concentration of lactate was at least 12 times higher than that used by Emery TABLE 5. VFA production from equimolar quantities of sodium lactate or sodium pyruvate incubated 8 hr with three types of ruminal ingesta a id Hay ingesta Silage ingestab Silage and grain ingestac Added Added Added Added Added Added lac- pyr- lac- pyr- lac- pyrtate uvate tate uvate tate uvate etate Propionate Butyrate Isovalerate. - Valerate... Downloaded from on April 2, 218 by guest a Represents the difference (expressed in milliequivalents per 1 ml) in VFA production between control flasks containing no lactate and experimental flasks containing lactate. b Cow's average daily consumption was 25 kg of an all-corn-silage ration. c Cow's daily consumption was 39 kg of corn silage + 4 kg of concentrate mixture. et al. Lactate metabolism followed zero-order kinetics with both the hay and grain ingesta, suggesting that these levels of lactate were in excess of the amount that could maximally be degraded by lactate-utilizing organisms. Under these conditions, polymerization would not be expected to have any effect on the rate of lactate utilization. An insufficient number of points on the curves (Fig. 1) obtained with the silage and lactate ingesta makes it impossible to say with certainty that lactate utilization followed first-order

6 VOL. 16, 1968 LACTATE METABOLISM BY RUMINAL INGESTA 685 TABLE 6. Effect ofph on VFA production from 5 ml of hay or grain? ingesta incubated 4 hr with 3.4 meq of sodium lactatea Ingesta Hay Grain ph etate Pro- Butyr- Isopionate ate vater Valerate a Expressed as milliequivalents per 1 ml per 4 hr. kinetics, but the suggestion is there. Even though the concentration of lactate was no different than for the hay or grain ingesta, the rate at which lactate was utilized was considerably faster, and it appeared to be proportional to its concentration. This is not an unexpected observation, for one would expect an enrichment of lactateutilizing organisms on these types of rations. Since no difference was observed between the rates of utilization of polymerized and nonpolymerized lactate under these conditions, one must conclude that 17% polymerized lactate is utilized as rapidly as the lactate monomer. The distribution of lactate carbon suggests that acetate was the principal metabolic end product with the hay, grain, and silage ingesta, whereas propionate contained the majority of label when the lactate ingesta were used. Other investigators, using ingesta from cows fed a variety of rations, observed that the major part of added '4C-lactate was converted to acetate (3, 8, 2). Baldwin et al. (3) made observations similar to those reported in this study. By incubating lactate-2-14c or -3-14C with five types of ingesta, they noted that the ratio of 14C-acetate to 14C-propionate ranged from 1.8 on an alfalfa hay ration to 2.5 on a grass silage-lactic acid-supplemented ration. Although acetate always contained the majority of label, the amount recovered in propionate increased as the availability of lactate increased. In contrast to these observations, Wallnofer et al. (28), who incubated lactate-2-14c with ingesta from three different rations, noted that 65% or more of the lactate carbon recovered in the VFA fraction was located in propionate. In much less definitive experiments, evidence has been gathered to suggest that propionate may be the major end product of lactate dissimilation in the rumen (13, 14, 17, 23, 26, 27). It is difficult to reconcile these apparent differences concerning the relative proportions of acetate and propionate formed from lactate in the rumen. At least seven bacterial genotypes capable of degrading lactate and known to inhabit the rumen have been described (1, 9, 1, 18, 19), but less than half of them have been found in large enough numbers to have a significant effect on ruminal metabolism. The variation in type of lactate utilizers, as well as their variation in number, the latter of which seems to be more variable between animals than for other classes of bacteria (21), may contribute to the apparent disparity in the observations. The literature contains insufficient information about the variation in numbers and types of ruminal lactate utilizers with different feeding regimens. Since no bacterial counts were made in this study, explanations offered on the basis of microbial variability are speculative. A partial explanation for reported differences in ruminal lactate metabolism is suggested by examining Tables 1 and 2. By use of the hay ingesta as an example, we noted that lactate dissimilation resulted in a net loss of acetate and a large increase in butyrate (Table 2). Propionate production was minimal. As mentioned earlier, the isotope study indicated that acetate was the principal end product of lactate degradation when hay ingesta were used. These results suggest that acetate is the immediate end product of lactate metabolism, but after release from the microbial cell and equilibration with the extracellular acetate pool, acetate is readmitted by the organism and used for butyrate synthesis. Depending upon the type of ration fed, one would predict from these observations that lactate in the ration or lactate produced in the ruminal fermentation would tend to lower the molar percentage of acetate, increase the molar percentage of propionate, and increase the molar percentage of butyrate. This phenomenon has, in fact, been observed or suggested in the majority of published studies dealing with lactate metabolism by ruminal contents either in vivo or in vitro (13, 14, 17, 22, 24, 26, 27) and in the unpublished studies of Laarman and Baumgardt and Bath and Rook (see 25). Phillipson (23) and Senel and Owen (25) noted that dietary lactates decreased the molar percentage of ruminal acetate, increased the molar percentage of propionate, and had no consistent effect on butyrate. Emery et al. (15) observed no difference in the relative amounts of ruminal VFA produced when lactate was included in the diet. Downloaded from on April 2, 218 by guest

7 686 SAlTER AND ESDALE APPL. MICROBIOL. The bulk of data is consistent with the view that both acetate and propionate are important metabolites of ruminal lactate, with propionate growing in importance as the amount of dietary starch or lactate increases. etate is not a terminal end product of lactate, however, but is used in the synthesis of butyrate. In some cases, this may result in a net decrease of acetate. Figure 2 shows the known metabolic pathways by which lactate can be metabolized by ruminal microorganisms. Propionate may be formed from lactate by the reductive route, involving the CoA esters of lactate, acrylate, and propionate, or by the randomizing route employing several dicarboxylic acids as intermediates. etate and butyrate are derived from lactate via pyruvate according to the scheme in Fig. 2. When lactate is oxidized to pyruvate, the formation of butyrate from extracellular acetate may be obligatory in that an electron sink must be provided for the two hydrogens generated in lactate oxidation to pyruvate. The data in Table 3 support the suggestion that extracellular acetate serves as an electron acceptor and that, through the formation of butyrate, an electron sink is provided for the pair of hydrogen atoms released in lactate oxidation to pyruvate. Co, The amount of acetate incorporated into butyrate was greatly enhanced when lactate was present, and, as was expected, the movement of butyrate carbon to acetate was reduced when lactate was present. Lactate has little if any effect on the randomization of propionate carbon. The electrode potential at which lactate is oxidized to pyruvate is too high for reduction of ferredoxin or similar low-potential carriers. Hydrogen is not formed, and therefore propionate, butyrate, or valerate production is an alternative for maintaining an oxidation-reduction balance. Apparently, butyrate production is favored. If the pair of hydrogens released in lactate oxidation to pyruvate is dictating the formation of butyrate to maintain an oxidation-reduction balance, one would expect pyruvate to yield mostly acetate and little or no butyrate. The Downloaded from results in Table 5 show that this is, in fact, what happens. With the use of the hay ingesta, the VFA production data, the metabolism of lactate-2-14c, and the randomization of ruminal VFA carbon suggest that acetate is the primary end product, with butyrate being the ultimate end product of lactate metabolism. Metabolism of pyruvate to ruvate -Oxaloacetate *kt- 2H Pr \2H.actate C Malate 1H2 /k.h2 on April 2, 218 by guest Crotonyl-CoA Xr2H ATP? (-Z2H Propionate.4- Butyryl-CoA \ZTP Butyrate FIG. 2. Outline of volatile fatty acidformation from lactate.

8 VOL. 16, 1968 LACTATE METABOLISM BY RUMINAL INGESTA 687 acetate yields two less hydrogens than does lactate, creating no need for butyrate formation. The reduced flavine or pyridine nucleotides generated in pyruvate metabolism to acetate are apparently utilized elsewhere. If ferredoxin is involved in the phosphoroclastic reaction, hydrogen gas may be formed (2). Hydrogen, in turn, could be used by methanogenic bacteria to produce methane. Randomization of ruminal VFA has been known for some time, particularly the extensive incorporation of extracellular acetate into butyrate (6, 16). That lactate greatly enhances butyrate formation from acetate has not been demonstrated before. The recent observation of Davis (12), which indicates that a low-roughage diet caused a greater incorporation of extracellular acetate into butyrate than did a mediumroughage diet, is consistent with the view that lactate enhances butyrate formation from acetate. Jayasuriya and Hungate (2) demonstrated that lactate is not an important intermediate in the rumen fermentation in hay-fed animals, but it may be an intermediate for as much as one-sixth of the total substrate of grain-fed animals. The presence of acetate appeared necessary for maximal utilization of lactate. Upon incomplete removal of acetate, lactate metabolism was partially impaired (Table 4). More complete removal of acetate by extensive washing greatly reduced lactate metabolism. Only small amounts of acetate were apparently needed for lactate metabolism, as evidenced by the need for thorough washing of the cells to obtain the effect. The conversion of some butyrate to acetate probably provided enough acetate to serve as an electron acceptor so lactate metabolism could proceed in the presence of butyrate. Bhat and Barker (7), working with Clostridium lactoacetophilum, later identified as C. butyricum (11), demonstrated that acetate was necessary for lactate utilization by that organism. Bryant and Burkey (11) found essentially the same requirement for C. tyrobutyricum, an organism isolated from silage. More definitive experiments must be done before one can say with assurance that acetate is necessary for lactate utilization by the mixed ruminal microflora. etate appears necessary for lactate utilization, and this observation is in agreement with findings concerning other anaerobic lactate utilizers. The effect of ph on lactate metabolism and VFA production was examined with both the hay and grain ingesta. Fermentation was markedly inhibited (Table 6), as evidenced by total VFA production, above ph 7.4 and below ph 6.2. More interestingly, there was a sizable shift in the relative amounts of acetate and butyrate within this ph range, even though total VFA production remained relatively constant. With both types of ingesta, acetate production was highest at ph 7.4, propionate at 6.8, and butyrate at 6.2. Between ph 6.2 and 7.4, propionate production did not change appreciably, but acetate and butyrate production was either increased or decreased by 5 to 1%. The reason for the shift is not clear. Butyrate formation probably occurs via reversal of the f-oxidation pathway. A hydrogen ion participates in the formation of,b-hydroxybutyrate from acetoacetyl coenzyme A. The increased hydrogen ion concentration may drive the reaction more in the direction of butyrate formation. There remains the possibility of differential activity among the microflora, resulting from the various ph levels. If this were true, however, one might have expected greater variation in the amount of propionate formed. A teleological explanation may be offered on the grounds that the microorganism synthesizes butyrate *from acetate when threatened by low acidity, thus reducing acidity 5%O by turning two acidic molecules into one. Perhaps the butyrate-synthesizing enzymes that are most active at the lower ph levels were selected because they offered a positive survival value. Nevertheless, the effect of ph on VFA production from ruminal ingesta containing lactate was quite striking. Some inconsistencies in the literature dealing with ruminal lactate metabolism may be due in part to the influence of ph. In this study, a uniform ph for all four types of ingesta was not maintained. The hay ingesta ranged from ph 6.4 to 6.8, the silage ingesta from 5.8 to 6.4, and the grain and lactate ingesta from about 5.5 to 6.4. It is doubtful, in this case, whether ph could account for but a small portion of the different patterns of VFA production observed with each type of ingesta. Propionate production remained relatively insensitive to ph, aside from the total inhibitory effect of either high or low ph (Table 6). Among types of ingesta, however, gross differences in propionate production were observed. Furthermore, butyrate production was maximal with the hay ingesta, which had the highest average ph, and was lowest with the grain and lactate ingesta, which had the lowest ph. This would not be expected on the basis of a ph effect. An attempt was made to construct crude carbon balances of lactate metabolism, but the technique employed in this study subjected these calculations to considerable variability. In addition, inadequate control of ph between the control incubation vessel and the lactate incubation vessel resulted in a small difference in ph between the two, with the control usually being slightly lower in ph. This was of little consequence for Downloaded from on April 2, 218 by guest

9 688 SATrER A,NID ESDALE APPL. MICROBIOL. the hay and silage ingesta, but for the grain and lactate ingesta the control vessel would be more vulnerableto a ph inhibitory effect. Consequently, the results in Table 1 are most accurately viewed in a qualitative sense rather than a stoichiometric sense. ACKNOWLEDGNIENTS This investigation was supported by the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. The authors acknowledge Nancy Rainey and Larry Ford for assistance in some of the laboratory analyses and Ralph Lance for the care provided to the animals. LITERATURE CITED 1. Annison, E. F., and D. Lewis Metabolism in the rumen, p. 48. John Wiley and Sons, Inc., New York. 2. Baldwin, R. L Physiology of digestion in the ruminant, p In R. W. Daugherty [ed.] Butterworth, Inc., Washington, D.C. 3. Baldwin, R. L., W. A. Wood, and R. S. Emery Conversion of lactate-c14 to propionate by the rumen microflora. J. Bacteriol. 83: Barker, S. B., and W. H. Summerson The colorimetric determination of lactic acid in biological material. J. Biol. Chem. 138: Baumgardt, B. R Practical observations on the quantitative analysis of free volatile fatty acids (VFA) in aqueous solutions by gasliquid chromatography. Univ. Wis. Dept. Dairy Sci. Dept. Bull Bergman. E. N., R. S. Reid, M. G. Murray, J. M. Brockway, and F. G. Whitelaw Interconversions and production of volatile fatty acids in the sheep rumen. Biochem. J. 97: Bhat, J. V., and H. A. Barker Clostridium lacto-acetophilum nov. spec. and the role of acetic acid in the butyric acid fermentaton of lactate. J. Bacteriol. 54: Bruno, C. F., and W. E. C. Moore Fate of lactic acid in rumen ingesta. J. Dairy Sci. 45: Bryant, M. P Bacterial species of the rumen. Bacteriol. Rev. 23: Bryant, M. P Symposium on microbial digestion in ruminants: Identification of groups of anaerobic bacteria active in the rumen. J. Animal Sci. 22: Bryant, M. P., and L. A. Burkey The characteristics of lactate-fermenting sporeforming anaerobes from silage. J. Bacteriol. 71: Davis, C. L etate production in the rumen of cows fed either control or low-fiber, high-grain diets. J. Dairy Sci. 5: Ekern, A., and J. T. Reid Efficiency of energy utilization by young cattle ingesting diets of hay, silage, and hay supplemented with lactic acid. J. Dairy Sci. 46: Elsden, S. R The fermentation of carbohydrates in the rumen of the sheep. J. Exptl. Biol. 22: Emery, R. S., J. W. Thomas, and L. D. Brown Fermentation, absorption, and feeding results with L(+) lactic acid monomer and polymer and DL-lactate salts. J. Animal Sci. 25: Gray, F. V., A. F. Pilgrim, H. J. Rodda, and R. A. Weller Fermentation in the rumen of the sheep. IV. The nature and origin of the volatile fatty acids in the rumen of the sheep. J. Exptl. Biol. 29: Hueter, F. G., G. L. Shaw, and R. N. Doetsch Absorption and dissimilation of lactate added to the bovine rumen and the resulting effects on blood glucose. J. Dairy Sci. 39: Hungate. R. E The rumen and its microbes, p. 76. ademic Press, Inc., New York. 19. Hungate, R. E., M. P. Bryant, and R. A. Mah The rumen bacteria and protozoa. Ann. Rev. Microbiol. 18: Jayasuriya, G. C. N., and R. E. Hungate Lactate conversions in the bovine rumen. Arch. Biochem. Biophys. 82: Kistner, A., and L. Gouws Bacteria of the ovine rumen. II. The functional groups fermenting carbohydrates and lactate on a diet of lucerne (Medicago sativa) hay. J. Agr. Sci. 59: Montgomery, M. J., L. H. Schultz, and B. R. Baumgardt Effect of intraruminal infusion of volatile fatty acids and lactic acid on voluntary hay intake. J. Dairy Sci. 46: Phillipson, A. T The fatty acids in the rumen of lambs fed on a flaked maize ration. Brit. J. Nutr. 6: Satter, L. D., J. W. Suttie, and B. R. Baumgardt Influence of accompanying substrate on in vitro volatile fatty acid formation from a- cellulose-c'4. J. Dairy Sci. 5: Senel, S. H., and F. G. Owen Relation of dietary acetate and lactates to dry matter intake and volatile fatty acid metabolism. J. Dairy Sci. 49: Waldo, D. R., and L. H. Schultz Lactic acid production in the rumen. J. Dairy Sci. 39: Waldo, D. R., and L. H. Schultz Blood and rumen changes following the intra-ruminal administration of glycogenic materials. J. Dairy Sci. 43: Wallnofer, P., R. L. Baldwin, and E. Stagno Conversion of C14-labeled substrates to volatile fatty acids by the rumen microbiota. Appl. Microbiol. 14: Wiseman, H. G., and H. M. Irwin Determination of organic acids in silage. J. Agr. Food Chem. 5: Downloaded from on April 2, 218 by guest