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Effect of Low-Roughage Diets on the Microflora and Lipid Metabolism in the Rumen

Changing the diet of five lactating cows and one nonlactating cow from high to low roughage induced milk fat depression in the lactating cows and altered the composition of the rumen microflora. While the numbers of lactic and propionic acid-producing bacteria increased, the numbers of Butyrivibrio spp. decreased. The numbers of lipolytic bacteria and the in vitro lipolytic activity of the rumen fluid were also decreased, as was the extent of hydrogenation of linoleic and linolenic acids combined in soybean oil incubated in vitro with rumen fluid. It is suggested that among the bacterial population in the rumen the vibrios, which were adversely affected by the low-roughage diets, may contribute significantly to both lipolysis and hydrogenation in the rumen.

Although most diets of ruminants normally contain high proportions of polyunsaturated acids as phospholipids and galactoglycerides, only small amounts of these acids are directly transferred to adipose tissue or to milk since most of them are metabolized to more saturated derivatives by the rumen microflora (31) before being absorbed from the small intestine. Hydrolysis of the complex dietary lipids is necessary before isomerization and hydrogenation of the fatty acids can take place (15). When cows are fed low-roughage diets, particularly those which depress milk fat secretion, the changes in the fatty acid composition of lipids of milk (25), blood plasma (9,21,27), digesta (29,33), and adipose tissue (30) suggest that hydrogenation in the rumen of dietary unsaturated acids may be reduced. Reductions in the numbers of ciliate protozoa have been associated with reduced hydrogenation (6), and lower numbers of these organisms and of the bacterium Butyrivibrio fibrisolvens, which also actively hydrogenates (23), are known to occur with lowroughage rations (4,18,24).
In this investigation the effect of low-roughage diets, including milk fat-depressing diets, on the composition of the microflora and the metabolism of feed lipids in the rumen have been examined more fully. The effect of these diets on the rumen fermentation are reported elsewhere (Latham, Sutton, and Sharpe, in press). MATERIALS AND METHODS Animals and their management. Rumen contents from four lactating Friesian cows with perma-nent rumen fistulas were used for in vitro studies on hydrogenation. Their diet was progressively changed from a control of high roughage (8 kg of hay and 10 kg of concentrate cubes, 20% dry weight as crude protein, daily) to a milk fat-depressing low-roughage diet in which flaked maize was substituted for part of the hay and concentrates until the milk fat content was depressed to less than 2%. The final proportions (in kilograms) of hay, concentrates, and flaked maize varied for each cow and were respectively 1, 1, 9 (cow G10); 1, 6, 4 (cow G9); 1, 6, 4 (cow B20) and 1, 1, 5 (cow G27).
Rumen contents from two more fistulated Friesian cows were used for in vitro studies on lipolysis. One (M21) in mid-lactation was treated similarly to the first four cows and received a final milk fat-depressing diet of 1, 2, 8 kg of hay, concentrates, and flaked maize, respectively, per day. The other cow (J), which was nonlactating, received an all-hay diet for 6 weeks and then a low-roughage diet of 20% hay and 80% flaked maize.
Sampling. Milk yields were recorded, and daily composite samples of the milk were analyzed for total fat content by the Gerber method (2) and for fatty acid composition by gas-liquid chromatography (26) with either a Perkin Elmer 880 or 900 model chromatograph. For characterizing the microflora, single samples of well mixed rumen digesta were taken from each of the lactating cows when given the high-and the low-roughage milk fat-depressing diets.
Characterization of the microflora. The Hungate (16) method of strict anaerobic culture as modified by Latham and Sharpe (17) was used throughout. All media for the enumeration of viable and lipolytic (tributyrin-hydrolyzing) bacteria and for the identification of isolates were based on medium 10 (4) as described by Latham, Sharpe and Sutton (18). Counts were made after 3 days of incubation, and the composition of the flora was determined by picking LATHAM, STORRY, AND SHARPE and subsequently identifying all the colonies (100-150) from suitable roll tubes used for the enumeration of the total viable population.
Direct microscopic counts of formalized suspensions of protozoa in glycerol-water (1:1, v/v) were made in a Neubauer counting chamber (Hawksley & Sons Ltd., Lancing, England), and differential counts of bacteria were made by the Breed technique (1) by using Gram-stained preparations. Lipolysis studies. Two methods were used for determining lipolytic activity.
(i) An agar diffusion method (19) was used to test the lipolytic activity of the bacteria from cow M21. The rate of hydrolysis of various triglycerides emulsified in buffered agar by test suspensions of bacterial cells was determined by measuring the diameter of the clear zones produced in the opaque gel at intervals during incubation at 30 C. Suitable suspensions of bacterial cells were obtained from 100 ml of rumen fluid which had been filtered through one layer of muslin. The filtered rumen fluid was centrifuged several times at approximately 300 x g for 5 to 10 min to remove the protozoa, and the supernatant fluid was centrifuged at 15,000 x g for 1 hr to sediment the bacteria. The bacteria were resuspended in 2.5 ml of 0.05 M sodium phosphate buffer, pH 7.0, and the amount of protein per milliliter of suspension was determined (20) so that in comparing the lipolytic activity between rations all values could be corrected to a unit value of 25 mg of protein per ml of suspension.
(ii) An incubation method was used to determine the lipolytic activity of whole rumen contents from cow M21 and of the protozoal, bacterial, and cell-free rumen fluid fractions of rumen contents from cow J when each cow was given highand low-roughage diets. Sediments obtained by centrifuging at 300 x g (rich in protozoa but contaminated with feed particles and large numbers of bacteria) and 15,000 x g (rich in bacteria) were resuspended in their original volume of cell-free (15,000 x g supernatant fluid) rumen fluid.
Duplicate flasks containing each of the rumen fluid fractions were incubated at 39 C in a water bath. Each flask was closed with a rubber bung adapted to gllow continuous stirring and gassing with 02-free nitrogen throughout the incubation. Emulsions of linseed oil or triolein were prepared by sonic oscillation (Dawe Soniprobe type 1130) in 0.05 M phosphate buffer, pH 7.0, containing 0.1% (w/v) gum acacia. The emulsion was purged with O2-free nitrogen, and 10% (v/v, final incubation mixture) added to one (experimental) of each pair of flasks to give 10 mM final concentration of triglyceride. The same volume of phosphate buffer containing only gum acacia was added to the remaining flasks (controls). After thorough mixing, 20-ml subsamples were removed from both the experimental and control flasks (0 hr), the reaction was stopped by acidifying to pH 2.0 with 50% HZSO4, and the sample was shell frozen with CO2-acetone. Additional subsamples were taken at 8 hr and treated similarly. All of the subsamples were then freeze-dried. Total lipid in the freeze-dried material was extracted in 2: 1 (v/v) chloroform-methanol, and the nonesterified fatty acids in the extract were separated by thin-layer chromatography (28) and estimated quantitatively by the colorimetric method of Duncombe (10).
Hydrogenation studies. Samples of rumen digesta were taken 4 to 5 hr after feeding from animals on the control diet and again from the same animals after they were established on the low-roughage diet and showing depressed milk fat. These samples were taken directly into each of two sterile graduated flasks and immediately gassed with 02-free nitrogen. Sufficient anaerobic dilution fluid (3) was added to dilute the contents by one-third to give a final volume of 1 liter. At the same time additional rumen samples were taken for microbiological analysis. The flasks were incubated in a similar manner to that used in the lipolysis studies. A 5-ml amount of 10% soybean oil emulsion ("Intralipid": Paines and Byrne, Greenford, England) was injected into the contents of one (experimental) of the flasks. After allowing 2 min for thorough mixing of the emulsion with the digesta, 30-g subsamples were taken from both the experimental and control flasks (0 hr), and again after 1, 2, 4, and 6 hr of incubation. The weight of each subsample was recorded, and the reaction was stopped by immediately transferring the sample into 2:1 (v/v) chloroform-methanol for extraction of total lipids. At each time of sampling, the pH and total numbers of bacteria were also recorded.
Extraction of total lipids and total fatty acids and determination of fatty acid composition. Total lipids from whole rumen contents in the hydrogenation studies and from freeze-dried rumen fluid fractions in the lipolysis studies were extracted in 2:1 (v/v) chloroform-methanol and washed with 0.88% (w/v) KCl solution as described by Folch, Lees, and Sloane Stanley (12). Total fatty acids were extracted from the total lipid samples after saponification, and their composition was determined by gas-liquid chromatography (26).

RESULTS
Milk fat. Although the composition of the low-roughage diets fed to individual animals differed, they -ll gave a marked depression in milk fat secretion. At the same time, the proportions of saturated fatty acids in the milk fat were decreased and the proportions of unsaturated fatty acids were increased (Table   1).
Rumen microflora. With the exception of cow G9, the low-roughage diets increased the number of viable bacteria in all cows by 0.6 to 1.6 log units but decreased the number of lipolytic (tributyrin-hydrolyzing) bacteria by up to 3 log units (Table 2). At the same time, these diets reduced by various amounts the numbers of ciliate protozoa. Of the two cows used in the lipolysis studies, M21 lost its ciliates, and there was a reduction in the number of ciliates in cow J which was not quantified. Only one (G10) of the four cows used in the hydrogenation studies lost its ciliates, while in cow G27 they were reduced by 90%. However, the two remaining cows (G9 and B20) lost only 32 and 45% of their ciliates, respectively.
The variation in the composition of the bacterial flora between cows given the lowroughage diets was considerable ( Table 3). The proportions of Selenomonas, Peptostreptococcus, Bifidobacterium, and Lactobacillus were particularly variable. Nevertheless, increases in lactogenic and propionogenic bacteria typical of low-roughage diets (4,18) were observed. The most consistent effect of the low-roughage diets on the bacterial flora was the severe reduction in the proportion of Butyrivibrio spp. Borrelia, which like Butyrivibrio is capable of hydrogenation (23,35), was also reduced.
Rumen lipolysis. Preparations of mixed rumen bacteria derived from cow M21 when fed the high-roughage diet hydrolyzed buffered emulsions of both tributyrin and tripalmitin, but tributyrin was hydrolyzed almost 2.5-fold as fast as tripalmitin (Fig. 1). On the low-roughage diet the rate of hydrolysis of both triglycerides by similar preparations of mixed rumen bacteria fell to one-fifth of the previously observed rates.
Incubations of whole rumen contents with emulsified linseed oil confirmed this apparent reduction in lipolytic activity (Table 4). Progressively less triglyceride was hydrolyzed as the roughage was reduced and the cereal content of the ration increased. In a separate experiment the lipolytic activity of the protozoal, bacterial, and cell-free rumen fluid fractions obtained from cow J given similar high-    and low-roughage rations were compared. The bacterial fraction associated with the hay diet had the greatest lipolytic activity, hydrolyzing 87.6% of the added triglyceride (Table 5), whereas the protozoal and cell-free rumen fluid fractions hydrolyzed only 28.6 and 13%, respectively. The same fractions from animals on the low-roughage ration hydrolyzed 5.8, 34.9, and 5.7%, respectively, indicating a considerable decrease in lipolysis by the bacterial fraction offset to some extent by a slight rise in that of the protozc level of unto increased N concentrate Rumen were fed th tions of tot digesta we when they These diff higher diet; age rations of rumen i were appre acids of di incubation any differe C18 fatty controlanm metabolisn The patt similar for mean resu diets are s Over the 6tion of lino form of so, diet, 59 and 63%, respectively, of that obtained on the high-roughage ration. The overall reduced rates of hydrogenation on the low-roughage diet were significant at the 5% and 1% level for linolenic and linoleic acids, respectively. The pattern of hydrogenation also differed between the two diets; with the high-roughage diet the hydrogenation appeared to commence soon after addition of the soybean oil and to progress rapidly up to the end of the fourth hour of incubation, whereas with the low-roughage ration the most rapid hydrogenation occurred between the second and sixth hours. )al fraction. In both experiments the study (18), the composition esterified fatty acid of dietary origin terial flora of nonlactating with increases in the proportion of and low-roughage diets es in the diet.
between cows fed the hydrogenation. When the cows present experiment the between-cow Le low-roughage diets, the concentraon the low-roughage ;al lipid and fatty acids in the rumen extended to the protozoa. ore two or three times greater than variability was not clear were fed the high-roughage diets. related to the high food erences can be accounted for by cows. ary intakes of lipid on the low-rough-It has been shown , and by increases in the contribution fats or oils are added microbial lipid (29). Although there rapidly adsorbed on food ,ciable amounts of oleic and linoleic ganisms (13,14); Czerkawski ietary origin present in the in vitro that levels of linseed oil systems, it has been assumed that ml of rumen fluid inhibited nces in the composition of the total Greater concentrations acids between samples from the this, while saturating i experimental incubations reflected rumen fluid (Demeyer, n of the added soybean oil. tion), have not been shown ;emns of hydrogenation obtained were In the present experiment, all four cows examined, and the total lipid and fatty lts for the high-and low-roughage used for the in vitro s hown in Fig. 2 and 3, respectively. lipolytic and hydrogenating *hr incubation period, the hydrogenagreater with the low-roughage lenic and linoleic acids added in the high-roughage diets, ybean oil was, on the low-roughage genation studies the    acids in "Intralipid" during incubation with-rumen contents from cows fed a low-roughage diet.
was added at the rate of 50 mg/100 ml of digesta, more than 800 mg/100 ml of digesta was added in the lipolysis studies. However, serial samples taken during the various incubation experiments revealed no major change in either the direct count or motility of bacteria and protozoa, indicating that even at the high substrate levels used in the lipolysis studies the added triglyceride had no apparent deleterious effect on the microflora. It is probable that the reduced lipolytic activity of the rumen digesta found with the low-roughage diets was related to the reduced numbers of lipolytic vibrios. These diets greatly reduced the viable counts of tributyrin-hydrolyzing bacteria, and approximately 70% of the tributyrinolytic bacteria isolated on a previous occasion in this laboratory from the same medium were found to resemble butyrivibrios.
Five out of 25 of these strains were able to hydrolyze long-chain triglycerides up to triolein. In the present work, the strongly lipolytic vibrio Anaerovibrio lipolytica was not isolated, and subsequent work in this laboratory suggests that this and similar lipolytic organisms constitute no more than 1% of the viable population. This effect of diet on lipolysis is in agreement with recent observations of Demeyer (personal communication) who found that the inclusion of sucrose in the diet ot'a sheep or of glucose in an in vitro incubation of rumen fluid reduced lipolysis.
It is not clear why the lipolytic activity of the protozoal fraction should have increased with the low-roughage diet. Clarke and Hawke (7) found that much of the lipolytic activity of this fraction could be transferred to the bacterial fraction after homogenization, and they therefore concluded that many lipolytic bacteria were attached to the particulate matter. Since on the low-roughage diet many starch granules will also sediment at the low "g" values used for separating the protozoal fraction of rumen fluid, they will, in so doing, bring with them even larger numbers of adhering bacteria among which may be actively lipolytic strains.
As indicated by the changes in milk fat composition and from the results of the in vitro incubations, the hydrogenating activity of the rumen contents was also reduced by the lowroughage rations. Strains of Butyrivibrio fibrisolvens were the first rumen bacteria to be implicated in the biohydrogenation of polyunsaturated fatty acids and were found to hydrogenate to the monoene (23). Since then laboratory strains of Borrelia (35), a gram-negative micrococcus (22), a strain each of Eubacterium and Ruminococcus (34), and two strains of cellulolytic Clostridium spp. (32) have all been found to biohydrogenate in a similar manner. However, only two strains of bacteria have so far been reported to be capable in pure culture of the complete reductive process to the saturated acid (34). Under normal conditions of high-roughage feeding, Butyrivibrio fibrisolvens is by far the most numerous of all of these biohydrogenating organisms found in the rumen and in the present experiment comprised 25.0% of the viable population in cows fed the high-roughage diets. On the assumption that the isolates of Butyrivibrio and Borrelia included hydrogenating strains, the reduction in their numbers which occurred with the lowroughage diets is consistent with the decrease in hydrogenation observed in vitro. Tove and Matrone (30) reported that, compared with conventional diets, the reduction of the monoenoic to the saturated acid proceeded more slowly with high-concentrate diets. As most workers have not been able to isolate strains which can carry out this final reductive step, such organisms are unlikely to occur in the majority flora, yet, like the butyrivibrios, they would appear to be greatly affected by dietary changes.
Although the protozoa-rich fraction of rumen contents has been shown to have biohydrogenating activity (5). the nule (l individual species of ciliate protozoa in this activity remains equivocal (31). However, studies on the plasma lipids of lambs with or without rumen ciliates indicate that dietary C,. unsaturated fatty acids are more effectively hydrogenated in the presence of ciliates (A. K. Lough, Proc. Nutr. Soc., 27:30A, 1968). It is therefore of interest that in the present work the reduction in hydrogenation induced by the low-roughage diets was no greater in the two cows which either lost or suffered a 90% reduction in the numbers of ciliates than in the remaining cows which retained between 55 and 70% of their ciliates. Because Hawke and Silcock (15) have recently shown that only nonesterified fatty acids can be hydrogenated, the present results suggest that, in addition to the adverse effects of low-roughage rations on the numbers of potential biohydrogenating organisms, a reduction in lipolysis may also contribute to the observed reduction in hydrogenation.

ACKNOWLEDGMENTS
The analysis of total fatty acid composition by A. J. Hall. the care of the animals by V. W. Johnson, and the capable technical assistance of Ann Siddell are gratefully acknowledged.