Bacteriat. Wooster, Ohio B835 (17), obtained from C. Henderson, Rowett Research

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1981, p /81/ $02.00/0 Vol. 42, No. 5 Effects of Long-Chain Fatty Acids on Growth of Rumen Bacteriat A. E. MACZULAK,lt B. A. DEHORITY,2* AND D. L. PALMQUIST' Departments of Dairy Science' and Animal Science,2 Ohio Agricultural Research and Development Center, Wooster, Ohio Received 1 April 1981/Accepted 13 August 1981 The effects of low concentrations of long-chain fatty acids (palmitic, stearic, oleic, and vaccenic) on the growth of seven species (13 strains) of rumen bacteria were investigated. Except for Bacteroides ruminicola and several strains of Butyrivibrio fibrisolvens, bacterial growth was not greatly affected by either palmitic or stearic acids. In contrast, growth of Selenomonas ruminantium, B. ruminicola, and one strain of B. fibrisolvens was stimulated by oleic acid, whereas the cellulolytic species were markedly inhibited by this acid. Vaccenic acid (trans All 18:1) had far less inhibitory effect on the cellulolytic species than oleic acid (cis A9 18:1). Inclusion of powdered cellulose in the medium appeared to reverse both inhibitory and stimulatory effects of added fatty acids. However, there was little carry-over effect observed when cells were transferred from a medium with fatty acids to one without. Considerable variation in response to added fatty acids was noted among five strains of B. fibrisolvens. In general, exogenous long-chain fatty acids appear to have little, if any, energy-sparing effect on the growth of rumen bacteria. Dietary fat is a useful energy source for dairy cows to meet the high-energy demand of milk production. However, when fat is included in ruminant diets, decreases in cellulose, crude fiber, and nitrogen-free extract digestibilities may occur (1, 7, 8, 23, 24). Long-chain fatty acids (LCFA) inhibit the growth of certain rumen and nonrumen bacteria (17, 20). Although the mechanism of inhibition is not certain, it has been ascribed to adsorption of LCFA onto the bacterial cell surface, thus impeding uptake of nutrients into the cell (15, 16, 18). Reversible adsorption of several LCFA onto rumen bacterial cells has been demonstrated (16, 19). In this study, the effects of LCFA on the growth of several species of rumen bacteria were investigated. Strain differences and possible mode of action were also studied. MATERLALS AND METHODS The bacteria used in this study were: Butyrivibrio fibrisolvens H1Ob, H17c, H4a, and D16f, Bacteroides ruminicola H8a, Lachnospira multiparus D15d, Ruminococcus flavefaciens B34b, and Bacteroides succinogenes B2la (9-11); Selenomonas ruminantium GA192, B. succinogenes S85, R. flavefaciens C94, and Ruminococcus albus 7 (2, 4, 5); and Butyrivibrio sp. t Approved for publication as journal article no of the Ohio Agricultural Research and Development Center. t Present address: Department of Animal Science, University of Kentucky, Lexington, KY B835 (17), obtained from C. Henderson, Rowett Research Institute, Aberdeen, Scotland. The characteristics of this latter strain were similar to the species B. fibrisolvens used in this study (10, 11). The composition of the complete-medium used for bacterial growth studies was similar to that of Scott and Dehority (22), except that the cation concentration in mineral mix B was reduced to 20% of normal. The medium was prepared by previously described procedures (11). The concentration of energy substrate (either glucose or cellobiose, depending on the organism studied) added to the basal mediumn was limiting, to detect any growth response due to the addition of LCFA. Limiting substrate concentrations were determined experimentally. Energy substrate and concentrations used for each strain were as follows: B. succinogenes B21a and S85 and B. ruminicola H8a-0.1% glucose; B. fibrisolvens HlOb and D16f, S. ruminantium GA192, L. multiparus D15d-0.167% glucose; Butyrivibrio sp. B % glucose; B. fibrisolvens H4a and H17c % glucose; and R. albus 7 and R. flavefaciens C94 and B34b-0.133% cellobiose. Cultures grown 15 to 20 h in complete medium were diluted 1:10 with anaerobic dilution solution (3) and used as inoculum at 0.1 ml per 7 ml of medium in culture tubes (16 by 150 mm). Duplicate cultures were incubated at 39 C, and optical density, measured at 600 nm with a Bausch & Lomb Spectronic 20, was used to estimate growth. The coefficient of variation for estimation of growth from duplicate tubes was 17.9%. There were no differences among microbial species or levels of added fatty acid on the precision of growth estimates. Purity of each LCFA, except vaccenic, was con- 856

2 VOL. 42, 1981 FATTY ACIDS AND RUMEN BACTERIAL GROWTH 857 firmed by gas chromatographic analysis as described by Palmquist and Conrad (21). Palmitic acid (C16:0) (Nutritional Biochemicals Corp., Cleveland, Ohio) was 98.5% pure. Stearic acid (C18:0) (Nutritional Biochemicals Corp.) and oleic acid (cis C18:1) (Sigma Chemical Co., St. Louis, Mo.) were greater than 99% pure. Capric acid (C10:0) (U.S. Biochemical Corp., Cleveland, Ohio) assayed 95% pure. Vaccenic acid (trans All1 18:1), 85% pure as supplied, was obtained from Calbiochem (Los Angeles, Calif.). Media used to determine the effects of LCFA on the growth of rumen bacteria were prepared by adding fatty acid in ethanol to the culture tube, evaporating the ethanol under nitrogen, anaerobically adding a 7- ml sample of medium, and autoclaving the individual tubes. The LCFA were added at concentrations ranging from to 0.02% of the medium. Control medium contained no fatty acid. To determine the effects of LCFA on growth in the presence of particulate matter, B. fibrisolvens H1Ob, L. multiparus D15d, and B. ruminicola H8a were grown in the presence of various levels of C16:0 or C18:1 with powdered cellulose (Solka-Flok BW-40; Brown Company, Berlin, N.H.) added at the same concentration (weight basis) as the highest level of LCFA. Control medium contained the powdered cellulose, but without added LCFA. RESULTS The effects of various levels of palmitic acid on the growth of the bacterial strains are shown in Table 1 and graphically for three strains in Fig. 1. Values are the means of duplicate tubes. Two of the three strictly gram-negative species, S. ruminantium and B. succinogenes, showed - only mild inhibition of growth, whereas the third, B. ruminicola, showed definite inhibition. Organism TABLE 1. Considerable variation in growth response was noted among the five strains of B. fibrisolvens, particularly at the highest concentration of palmitic acid (0.02%). Some discrepancies can be noted in these data, i.e., slightly greater inhibition of strain H4a at 0.001% palmitate concentration than at or 0.01%. No obvious explanation can be offered, except for normal biological variation. The second replicate for this strain did not show a similar decrease at 0.001%. Changes of less than 10% can probably be considered of doubtful significance. The remaining species, all gram positive or gram variable, showed little inhibition, except for R. flavefaciens C94 at 0.01%. The presence of the LCFA appeared to affect either the rate of growth, lag phase, or both, for several strains (Fig. 1). Stearic acid had little effect on the growth of most organisms (Table 2). L. multiparus and B. fibrisolvens H17c showed moderate inhibition at the higher levels, whereas marked inhibition was noted for Butyrivibrio sp. B835. A markedly different type of response was noted with the addition of oleic acid, an unsaturated LCFA (Table 3 and Fig. 2).The two gramnegative species, S. ruminantium and B. ruminicola, showed no change or an increase in growth (optical density) at all levels tested. Growth of Butyrivibrio sp. B835 was also increased. L. multiparus, a weakly gram-positive organism, and three strains of B. fibrisolvens were mildly inhibited at higher concentrations. Growth of the gram-positive or gram-variable cellulolytic ruminococci was almost completely inhibited at oleic acid concentrations of %. Effects ofpalmitic acid on the growth of rumen bacterial strains Growth' with C16:0 in the medium at (%): B. fibrisolvens HlOb 31 (24)b 80.6 (27) 48.4 (27) 54.8 (27) 54.8 (30) D16f 56 (30) 98.2 (43) (43) 92.8 (47) 85.7 (49) H17c 33 (20) 84.8 (25) (28) 69.7 (31) 24.2 (28) H4a 43 (31) 72.1 (34) 88.4 (31) 83.7 (37) 23.2 (34) B (53) 98.3 (53) 95.0 (53) S. ruminantium GA (8) 93.7 (8) 95.2 (8) 87.3 (8) 79.4 (8) B. ruminicola H8a 60 (19) 75.0 (19) 50.0 (22) 55.0 (22) L. multiparus D15d 57 (21) (21) (21) (25) 94.7 (21) B. succinogenes S85 80 (36) (40) 98.7 (36) 92.5 (46) 82.5 (58) B21a 71 (48) (48) (48) 95.8 (59) 85.9 (95) R. albus 7 51 (15) 90.2 (15) 92.2 (15) 94.1 (15) 88.2 (15) R. flavefaciens B34b 32 (22) 87.5 (22) 96.9 (22) (22) 84.4 (25) C94 26 (40) (40) (40) 92.3 (47) 69.2 (59) a For medium without added fatty acid, growth is reported as maximum optical density (600 nm) x 100. Growth in medium with added LCFA is expressed as percentage of control maximum optical density. Values are means of duplicate tubes and are representative data from two or three replicates. b Numbers in parentheses indicate number of hours of incubation to reach maximum optical density.

3 858 MACZULAK, DEHORITY, AND PALMQUIST It was also of interest that the growth of B. fibrisolvens H17c was markedly inhibited, since this strain possesses considerable cellulolytic activity. For R. albus 7, the extent of growth was APPL. ENVIRON. MICROBIOL. not affected by the addition of % oleic acid; however, a marked increase was noted in the lag phase (Fig. 2). Vaccenic acid (trans-i1-octadecenoic acid) E 0.6 '0 g 0.5 o.0 0 OA A.-c R.% "O0.0% C 16: %.0.005%.*9.'1.' % I I I Ia Downloaded from Hours FIG. 1. Effects ofpalmitic acid on the growth of certain rumen bacteria. TABLE 2. Effects of stearic acid on the growth of rumen bacterial strains Growth' with C18:0 in the medium at (%): Organism B. fibrisolvens HlOb 43 (19)b 95.3 (19) 93.0 (19) 88.4 (19) 86.0 (19) D16f 84 (16) 97.6 (19) 91.7 (19) 89.3 (22) 83.3 (22) H17c 45 (12) 77.8 (15) 80.0 (15) 71.1 (15) 66.7 (19) H4a 111 (60) 94.6 (63) 90.1 (60) 91.9 (63) 85.6 (63) B (53) (53) 39.6 (34) 29.3 (34) S. ruminantium GA (8) 90.7 (8) 86.2 (8) 86.2 (8) 87.7 (8) B. ruminicola H8a 63 (17) 93.6 (17) 79.4 (19) 77.8 (17) 84.1 (17) L. multiparus D15d 71 (18) 87.3 (18) 87.3 (21) 70.4 (21) 71.8 (21) B. succinogenes S85 70 (36) (36) 92.8 (36) 97.1 (36) 98.6 (36) R. albus 7 50 (23) (23) 96.0 (23) 98.0 (23) 90.0 (23) R. flavefaciens B34b 18 (24) 88.9 (34) (34) (34) 88.9 (34) C94 19 (45) 89.5 (45) 84.2 (52) 94.7 (45) 78.9 (45) a For medium without added fatty acid, growth is reported as maximum optical density (600 nm) x 100. Growth in medium with added LCFA is expressed as percentage of control maximum optical density. Values are means of duplicate tubes and are representative data from two or three replicates. b Numbers in parentheses indicate number of hours of incubation to reach maximum optical density. on December 25, 2018 by guest

4 VOL. 42, 1981 FATTY ACIDS AND RUMEN BACTERIAL GROWTH 859 TABLE 3. Effects of oleic acid on growth of rumen bacterial strains Growtha with oleic acid (C18:1) in the medium at (%): Organism B. fibrisolvens HlOb 42 (16)b (20) (20) 66.7 (38) D16f 66 (20) (20) 89.4 (29) 87.9 (24) H17c 75 (70) 60.0 (84) 0 (100) 0 (100) H4a 85 (44) 68.2 (75) 80.0 (166) 98.8 (140) B (53) 94.7 (63) (92) (107) S. ruminantium GA (12) (12) (12) (12) B. ruminicola H8a 90 (16) (16) (20) (20) L. multiparus D15d 82 (18) 81.7 (21) 82.9 (24) 78.0 (24) R. albus 7 48 (23) (47) 18.7 (80) 14.6 (80) R. flavefaciens B34b 21 (37) 90.5 (57) 0 (57) 0 (57) C94 31 (40) 0 (120) 0 (120) 0 (120) a For medium without added fatty acid, growth is reported as maximum optical density (600 nm) x 100. Growth in medium with added LCFA is expressed as percentage of control maximum optical density. Values are means of duplicate tubes and are representative data from two or three replicates. b Numbers ih parentheses indicate number of hours of incubation to reach maximum optical density. O.5 B. fibrisolvens H 10 b ~~~~~~~~~~0 E E B. ruminicola H8a a0 OS C 18:1 * % u118p8 1OO1% 0. Hours FIG. 2. Effects of oleic acid on the growth of certain rumen bacteria. had little effect on the growth of B. fibrisolvens D16f, S. ruminantium GA192, or L. multiparus D15d; however, when added at a 0.01% concentration, growth of R. albus and R. flavefaciens was inhibited by 19 and 39%, respectively. The trans 18:1 acid thus had a far less inhibitory effect on the growth of the ruminococci than did cis 18:1 (oleic acid). The effects of capric acid on the growth of three strains of B. fibrisolvens (HIOb, D16f, and

5 860 MACZULAK, DEHORITY, AND PALMQUIST B835) were also determined. Definite growth enhancement was observed for all strains with 0.01% capric acid in the medium, ranging from 113 to 117% of controls. Possible carry-over effects of LCFA on the growth of several strains of bacteria were investigated by growing inoculum cultures in the presence and absence of LCFA. B. fibrisolvens D16f, S. ruminantium GA192, L. multiparus D15d, and R. albus 7 showed little or no growth inhibition in LCFA-free medium after the inoculum had been grown in medium containing 0.005% palmitic acid or 0.001% oleic acid. A slight cumulative effect, 20% decrease in growth, was observed when L. multiparus inoculum was grown in the presence of 0.005% palmitic acid and used to inoculate 0.005% palmitic acid medium. Growth of R. albus 7 decreased 35% after growth and transfer in medium containing 0.001% oleic acid. In all other instances, no cumulative effects were noted. To determine the possible effects of LCFA on bacterial growth in the presence of fibrous particulate matter, three non-cellulolytic strains were grown in media with various concentrations of LCFA, with and without added cellulose (100 mesh). The cellulose was added on a weight basis, equivalent to the highest concentration of LCFA. For B. fibrisolvens HlOb and B. ruminicola H8a, the extent of inhibition was markedly reduced (Table 4). This was particularly true for B. ruminicola H8a, in which 0.01% palmitic acid decreased growth by 10.6% in the presence of cellulose, whereas one tenth of that concentration, 0.001%, decreased growth by 49.2% in the absence of the cellulose. The growth of L. multiparus D15d was not greatly inhibited by palmitic acid and, quite unexpectedly, growth was slightly inhibited with cellulose in the medium. Similar results were obtained with oleic acid, i.e., added cellulose decreased growth inhibition in the strains inhibited by oleic acid and reduced stimulatory effects in those strains showing increased growth in the presence of oleic acid. TABLE 4. APPL. ENVIRON. MICROBIOL. DISCUSSION The limiting substrate conditions used in these growth studies suggest that exogenous LCFA have little, if any, energy-sparing effect on the growth of rumen bacteria. Although several instances of growth stimulation were observed with added LCFA, the magnitude of stimulation was relatively small. A growth response of about 127% was reported by Henderson (17) for Butyrivibrio sp. B835 in medium containing 0.001% oleic acid. In the present study, growth of the same organism, at the same oleic acid concentration, was 121% of control. However, substrate level was nonlimiting (0.6%) in Henderson's study as compared to an experimentally determined limiting level (0.2%) in this work. Similar results were obtained for this strain with capric acid. Sparing of energy required for de novo synthesis of fatty acids by direct incorporation of LCFA into celluar lipids should be more evident in a limiting substrate system. The three species of strictly gram-negative bacteria, S. ruminantium, B. ruminicola, and B. succinogenes, were relatively unaffected by inclusion of LCFA in the medium. The only exception to this was decreased growth by B. ruminicola in the presence of palmitic acid. Four strains of B. fibrisolvens, also described as a gram-negative species, were included in the present study, and marked strain differences were noted when growth took place in the presence 6f the different LCFA. Since none of the strains exhibited a growth stimulation to added oleic acid, as reported by Henderson (17), his strain, Butyrivibrio sp. B835, was obtained and tested under our conditions. Palmitic acid was much less inhibitory, and stearic acid was more inhibitory than found by Henderson for this strain. However, a similar enhancement of growth in the presence of low levels of oleic acid (0.001%) was observed. For all five strains, growth in the presence of 0.001% oleic acid ranged from 0% for H17c to 121% of controls for B835. Henderson Effect ofparticulate matter on growth of rumen bacteria in the presence ofpalmitic acid Growth' with C16:0 in the medium at (%): Organism _b B. fibrisolvens HlOb 44 (22)' 43 (13) 61.4 (33) 95.3 (16) 52.3 (30) 81.4 (16) 45.4 (30) 62.8 (10) L. multiparus D15d 57 (21) 69 (16) (21) 88.4 (16) (25) 82.6 (20) 94.7 (21) 58 (20) B. ruminicola H8a 65 (17) 66 (34) 50.8 (19) 97.0 (34) 33.8 (19) 98.4 (34) 36.9 (19) 89.4 (34) "For medium without added fatty acid, growth is reported as maximum optical density (600 nm) x 100. Growth in medium with added LCFA is expressed as percentage of control maximum optical density. Values are means of duplicate tubes. b, Without 0.01% cellulose; +, with 0.01% cellulose. 'Numbers in parentheses indicate number of hours of incubation required to reach maximum optical density.

6 VOL. 42, 1981 (17) also observed growth stimulation of strain B835 with capric acid. This was repeated in our laboratory, and a similar response was noted for our strains HlOb and D16f. The two latter strains were different from B835 and each other, when grown in the presence of oleic acid. Based on the data from all five strains of Butyrivibrio, it appears that large strain differences can occur. The Ruminococcus strain studied by Henderson (17), described as gram variable, was inhibited by stearic, palmitic, and oleic acids in an increasing order. Of the three Ruminococcus strains in this study, described as gram variable to gram positve, R. flavefaciens C94 was the only one inhibited to any extent by palmitic and stearic acids. All strains were markedly inhibited by much lower levels of oleic acid than Henderson's strain. Thus strain and species differences were also apparent in the genus Ruminococcus. Although the Gram stain classifies B. fibrisolvens as gram negative, electron microscopy has recently revealed it to have a gram-positve-type cell wall (6). This might explain its difference in growth response to the various LCFA from that of strictly gram-negative species. However, L. multiparus, which stains weakly gram positive, was not markedly affected by any of the LCFA. Previous studies have suggested that the growth of gram-positive bacteria is inhibited by LCFA to a greater extent than is that of gram-negative bacteria (14, 15, 17, 20); however, the present results with rumen bacteria would not completely support this conclusion. The cis-trans configuration of LCFA appears to influence the degree of inhibition or stimulation, since cis A9 18:1 was far more inhibitory than trans All 18:1. Demeyer and Henderickx have reported the cis-unsaturated C18 fatty acid to be more toxic to mixed rumen bacteria than the trans isomer (12). A possible explanation for this difference was suggested by the recent studies of T. C. Jenkins and D. L. Palmquist (submitted for publication), where they found that the inhibitory effects of LCFA on cell wall digestibility were inversely related to their ability to form insoluble calcium soaps. The order for completeness of soap formation in a mixed culture in vitro fermentation was stearate > vaccenate > palmitate > oleate. Even though calcium concentration was decreased in the basal medium for this study, enough was still present to complex about 300 mg of LCFA per liter of medium, which exceeded the maximum concentration of added LCFA (0.02%). Thus, the relative inhibitory effects of the different fatty acids may be partially explained by their selective removal from the medium as insoluble soaps. FATTY ACIDS AND RUMEN BACTERIAL GROWTH 861 Addition of cellulose to LCFA-containing culture media reduced both the inhibitory and stimulatory actions of C16:0 and C18:1, respectively, with three strains of non-cellulolytic bacteria. These observations are consistant with the theory that feed particles in the rumen compete with bacteria for adsorption of LCFA (16). Results from other studies have suggested that fat in the rumen preferentially adheres to fibrous plant particles rather than bacterial cells (13, 15). This apparently alleviates the cell surface interactions with LCFA and allows normal nutrient uptake by the microbes. The marked growth inhibition by oleic acid of the cellulolytic strains (B. fibrisolvens H17c, R. albus 7, and R. flavefaciens B34b and C94) is very interesting. If the cellulase enzyme is cell surface bound, adsorption of the LCFA either on the cell or substrate would probably interfere with the digestion of insoluble cellulose. However, the almost complete growth inhibition of these strains with a soluble substrate might suggest something different about the cell surface of cellulolytic species. If true, there appears to be some degree of specificity toward oleic acid (cis A9 18:1), since vaccenic acid (trans All 18: 1) was far less inhibitory. These studies indicate that for some species either the rate of growth, lag phase, or both were affected by additions of LCFA, even when maximum growth was not decreased. Assuming that LCFA interfere with cell nutrient uptake (14, 16, 17, 20), one or both of these parameters could be affected. In this study, data are presented in terms of hours required to reach maximum absorbance, which measures the length of the lag phase plus the rate of growth through the logarithmic phase. As the logarithmic phase commences, the ratio of fatty acid to bacterial cells decreases, so LCFA may be expected to have less effect on logarithmic growth and maximum absorbance than on growth in the lag phase. This appeared to be the case in the present experiments (Fig. 1 and 2), except at the high LCFA concentrations. If dietary fat exerts similar inhibitory effects on the bacterial population within the rumen, it is possible that with prolonged feeding, changes in the proportion of rumen bacterial species will take place. LITERATURE CITED 1. Brooks, C. C., G. B. Garner, C. W. Gehrke, M. E. Muher, and W. H. Pfander The effect of added fat on the digestion of cellulose and protein by ovine rumen microorganisms. J. Anim. Sci. 13: Bryant, M. P The characteristics of strains of Selenomonas isolated from bovine rumen contents. J. Bacteriol. 72:

7 862 MACZULAK, DEHORITY, AND PALMQUIST 3. Bryant, M. P., and L. A. Burkey Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36: Bryant, M. P., and R. N. Doetsch A study of actively cellulolytic rod-shaped bacteria of the bovine rumen. J. Dairy Sci. 37: Bryant, M. P., N. Small, C. Bouma, and I. M. Robinson Characteristics of ruminal anaerobic cellulolytic cocci and Cillobacterium cellulosolvens n. sp. J. Bacteriol. 76: Cheng, K.-J., and J. W. Costerton Ultrastructure of Butyrivibrio fibrisolvens: a gram-positive bacterium? J. Bacteriol. 129: Czerkawski, J. W The effect on digestion in the rumen of a gradual increase in the content of fatty acids in the diet of sheep. Br. J. Nutr. 20: Czerkawski, J. W., K. L. Blaxter, and F. W. Wainman The effect of linseed oil and linseed oil fatty acids incorporated in the diet on the metabolism of sheep. Br. J. Nutr. 20: Dehority, B. A Isolation and characterization of several cellulolytic bacteria from in vitro rumen fermentations. J. Dairy Sci. 46: Dehority, B. A Characterization of several bovine rumen bacteria isolated with a xylan medium. J. Bacteriol. 91: Dehority, B. A Pectin-fermenting bacteria isolated from the bovine rumen. J. Bacteriol. 99: Demeyer, D. I., and H. K. Henderickx The effect of C18 unsaturated fatty acids on methane production in vitro by mixed rumen bacteria. Biochim. Biophys. Acta 137: Devendra, C., and D. Lewis The interaction between dietary lipids and fiber in the sheep. Anim. Proc. 19: Galbraith, H., and T. B. Miller Effect of long APPL. ENVIRON. MICROBIOL. chain fatty acids on bacterial respiration and amino acid uptake. J. Appl. Bacteriol. 36: Galbraith, H., T. B. Miller, A. M. Paton, and J. K. Thompson Antibacterial activity of long chain fatty acids and the reversal with calcium, magnesium, ergocalciferol and cholesterol. J. Appl. Bacteriol. 34: Harfoot, C. G., M. L. Crouchman, R. C. Noble, and J. H. Moore Competition between food particles and rumen bacteria in the uptake of long-chain fatty acids and triglycerides. J. Appl. Bacteriol. 37: Henderson, C The effects of fatty acids on pure cultures of rumen bacteria. J. Agric. Sci. 81: Kodicek, E., and A. N. Worden The effect of unsaturated fatty acids on Lactobacillus helveticus and other gram-positive microorganisms. Biochem. J. 39: Maxcy, R. B., and C. W. Dill Adsorption of free fatty acids on cells of certain microorganisms. J. Dairy Sci. 50: Nieman, C Influence of trace amounts of fatty acids on the growth of microorganisms. Bacteriol. Rev. 18: Palmquist, D. L., and H. R. Conrad High fat rations for dairy cows. Effects on feed intake, milk and fat production, and plasma metabolites. J. Dairy Sci. 61: Scott, H. W., and B. A. Dehority Vitamin requirements of several cellulolytic rumen bacteria. J. Bacteriol. 89: Steele, W., and J. H. Moore The digestibility coefficients of myristic, palmitic and stearic acids in the diet of sheep. J. Dairy Res. 35: White, T. W., R. B. Grainger, F. H. Baker, and J. W. Stroud Effect of supplemental fat on digestion and the ruminal calcium requirement of sheep. J. Anim. Sci. 17: Downloaded from on December 25, 2018 by guest