Metabolism of Long-Chain Fatty Acids in the Rumen

Metabolism of Long-Chain Fatty Acids in the Rumen

Metabolism of Long-Chain Fatty Acids in the Rumen ROMANO VIVIANI Institute of Biochemistry, Faculty of Veterinary Medicine,1 Bologna, Italy I. Uni...

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Metabolism of Long-Chain Fatty Acids in the Rumen ROMANO VIVIANI Institute

of Biochemistry,

Faculty of Veterinary Medicine,1 Bologna, Italy

I.

University

Introduction A. General B. T h e Ruminants C. Methods Used for the Study of Long-Chain Fatty Acids in the Rumen II. Naturally Occurring Fatty Acids in Actual and Potential Feeds A. Microorganisms B. Cormophyta C. Animals III. Fatty Acid Composition of Rumen Microorganisms A. Bacteria B. Fungi C. Protozoa IV. Fatty Acid Metabolism of Rumen Microorganisms A. Lipid Hydrolysis and Absorption or Adsorption of Fatty Acids B. Biosynthesis C. Biohydrogenationof Unsaturated Fatty Acids D. Other Processes E. Metabolic Control V. Relationships between Long-Chain Fatty Acids and Some Metabolic Processes of Rumen Microorganisms A. Long-Chain Fatty Acids and Cellulose Digestion . . . . B. Long-Chain Fatty Acids and Methane Production . . . . C. Unsaturated Fatty Acids and Other Reductive Processes VI. Fate of Rumen Long-Chain Fatty Acids A. Absorption from the Digestive Tract B. Utilization for Ruminant Lipids

of

Bologna,

268 268 269 271 274 274 275 280 280 280 289 289 290 290 294 302 317 319

320 320 321 323 324 324 325

'The experiments by the author and his co-workers reported in this review were sup­ ported by grants from the "Consiglio Nazionale delle Ricerche (Roma, Italia)" for the "Gruppo di lavoro sulla fisiopatologia del rumine."

267

268

ROMANO VIVIANI VII.

VIII.

IX.

Effect of Rumen Metabolism on the Fatty Acid Composition of Ruminant Lipids A. Effects of Rumen Function Development B. Effects of Dietary Conditions Long-Chain Fatty Acids in Rumen Dysfunction A. Bloat B. Parakeratosis Conclusions References

I. A.

329 329 333 336 336 337 339 340

Introduction

GENERAL

The study of long-chain fatty acid metabolism in rumen is currently a very interesting subject: not only does it contribute to the under­ standing of normal and pathological lipid metabolism in the ruminant, but it can also aid in understanding problems in connection with rum­ inant growth and development and with foods from ruminants (meat, milk, etc.). Interest in the chemical nature and biochemical significance of the fatty acids of ruminant lipids dates to when the presence of "trans" isomers in depot fats was first observed (Bertram, 1928), and to the hypothesis advanced by Banks and Hilditch (1931) that the "stearic rich" depot fat depends on hydrogenation in the tissues. New atten­ tion was given to this subject when branched-chain fatty acids of the iso and anteiso types were discovered (Hansen and Shorland, 1951) and when it was observed that the ruminal content has the ability to hydrogenate unsaturated fatty acids (Reiser, 1951). The demonstration in the last decade that rumen contains large amounts of stearic acid, of odd- and branched-chain fatty acids (Garton, 1961; Keeney et al., 1962a; Viviani et al., 1963a), and of geometric and positional isomers of 18-carbon unsaturated fatty acids (Shorland et al., 1957) clearly indicates that lipid metabolism in ruminants is affected by the rumen microflora. In fact, if we consider that in 1 gm of fresh rumen content there are 100 billion bacteria and one million protozoa, and that the microbial species can vary according to the dietary conditions (Annison and Lewis, 1959, p. 22; Barnett and Reid, 1961, p. 16; Hungate, 1966, pp. 8, 92), it is easily understood how dietary lipids can undergo substan­ tial modifications before passing to the intestine for further utilization. A large proportion of the 142 acids that have b e e n identified in milk

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(Jensen et ah, 1967) d e p e n d upon biochemical processes in the rumen. In this review, an attempt is made to summarize the information now available on the subject of long-chain fatty acid metabolism in the rumen. This subject is, of course, really part of the larger topic of rumi­ nant lipid metabolism. For certain problems this review reports data accepted by various investigators, and referred to in the reviews by Garton (1960, 1961, 1964,1965) and Tove (1965) on lipid metabolism in the ruminant, and in the books on rumen by Annison and Lewis (1959, p. 155), Barnett and Reid (1961, p. 67), and Hungate (1966, p. 206). As the amount of published data in certain areas is as yet not large, new data, including our own work in progress, are introduced here. This report may be considered an attempt to formulate the problems and to indicate also where additional data are needed. B. T H E

RUMINANTS

In the biological evolution of animals, the great development of herbivores coincided with the change in pattern of climate and with the development of the grasses during the Tertiary period. In the Perissodactyla, the dominant group of herbivores of the Eocene period, there are now only 6 living genera, of a total of 158 known genera; in the Artiodactyla, which assumed dominance in postOligocene period, a similar proportion holds for the nonruminants whereas there are 73 living genera of ruminants (Simpson, 1945). A phenomenon that may have b e e n important in this evolutionary pro­ cess and in the greater resistance of ruminants can also be linked to the type of digestive apparatus and to nutritional utilization of vegeta­ bles. All herbivorous animals have an expanded part in the alimentary tract where vegetable fibrous food undergoes extensive fermentation before utilization. In this respect, the herbivores are divided into two major groups: herbivores presenting their ingested food for fermenta­ tion prior to gastric digestion, such as the true ruminants and the other ruminantlike animals; and those in which fermentation occurs in the hindgut. According to the present classifications, Ruminantia are dis­ tinguished from Tylopoda and Suiformes, and all three together con­ stitute the Artiodactyla. Rumen is present both in ruminants (ox, sheep, goat, deer, reindeer, giraffe, yak, buffalo, etc.) and in Tylopoda (camel, alpaca), which on the basis of anatomical characteristics are defined as pseudo-ruminants; other animals, belonging to the Sui­ formes, such as hippopotamus, can be considered ruminant-like.

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Among herbivores having a digestive apparatus expanded beyond the stomach, there are, in addition to Perissodactyla, also Proboscidata and several rodents. The stomach of ruminants consists of four compartments: rumen, reticulum, omasum, and abomasum. The compartments develop from the embryonic stomach and are relatively small in the newborn an­ imal and do not develop if the animal continues to be fed a milk diet exclusively. The presence of solid food, forage, and concentrates pro­ motes the anatomical and functional development of rumen, mainly by making it possible for a population of bacteria and protozoa to be­ come established in the rumen. These microorganisms determine biochemical processes on the diet, and the terminal products of fer­ mentation—volatile fatty acids (VFA) — stimulate the development of the ruminal mucosa and of the functionality of rumen. It was demon­ strated that volatile fatty acids stimulate biosynthesis of mRNA in the ruminal mucosa (Viviani et al., 1967a) and activate physiological pro­ cesses related to the rumination (McGilliard et al., 1965; Beghelli et al, 1967). Adult ruminants differ from other animals in that they can utilize enormous quantities of roughage. Such utilization is almost entirely dependent upon the microbial reactions that take place in the forestomach. The products of cellulolytic breakdown, apart from gases, are bacterial and protozoal polysaccharides and volatile fatty acids. The latter are absorbed by the animal from the rumen and omasum, while polysaccharides are utilized by the action of the ordinary animal digestive processes. In addition, the microbial population is capable of utilizing nonprotein nitrogen for the synthesis of amino acids and pro­ teins. The synthetic activity of microorganisms increases manyfold the vitamin content of the ingesta: this makes the animal virtually independent of dietary sources of all the vitamins except A, D, and E (Annison and Lewis, 1959, p. 14; Barnett and Reid, 1961, p. 2; Hungate, 1966, p. 331). Lipids, too, undergo notable modifications; in fact, lipids absorbed by intestine (Section VI, A) or those present in rumen, differ from ali­ mentary lipids (Sections II, B and III, A, C). In rumen are synthesized de novo even, odd, branched, saturated, and unsaturated fatty acids (Section IV, B); polyunsaturated acids are synthesized by protozoans, monounsaturated acids by bacteria (Section IV, B). Furthermore, biohydrogenation of unsaturated fatty acids occurs in rumen (Section IV, C). Adult ruminants do not require either essential amino acids and vi­ tamins of the B group or essential fatty acids (EFA), since these com-

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pounds are synthesized by the ruminal micropopulation. T h e same compounds are essential, however, for the young ruminant fed a milk diet, which in this case behaves like a monogastric animal (Section VII, A). The biochemical features that distinguish ruminants from the other Artiodactyla and other nonruminant herbivores (such as, Perissodactyla and Rodentia), in addition to low levels of blood glucose and the presence of volatile fatty acids in plasma, are the high content of stearic acid in depot fats and the presence of characteristic fatty acids in milk and in tissue lipids (Section VI, B).

C.

M E T H O D S U S E D FOR THE STUDY O F LONG-CHAIN A C I D S IN T H E R U M E N

FATTY

1. General Long-chain fatty acid metabolism may be studied by the general techniques used for biochemical reactions in rumen. Basic principles and literature thereon are reported in the classical books by Barnett and Reid (1961, p. 2) and Annison and Lewis (1959, p. 55). Experimentation may be carried out either in vivo or in vitro; the animals used are ovines or bovines with a permanent ruminal fistula, fed various diets: for example, purified and lipid-free diets for evalua­ tion of de novo biosynthesis (Viviani and Lenaz, 1963), purified diets containing lipids (Tove and Matrone, 1962), practical diets with high fiber or concentrates content (Section VII, B). The ruminal content, used as such or divided into fractions, has b e e n compared with fatty acid content of other parts of the digestive apparatus, and absorption phenomena have b e e n considered (McCarthy, 1962a,b; Felinsky et al, 1964; Ward et al, 1964). The rumen reticulum is similar to a continuous-feed system, in which fresh feed and saliva mix with the fermenting mass in a vat, and fluid and feed residues leave in quantities equivalent to those en­ tering (Hungate, 1966, p. 368). The in vivo fatty acid metabolism comprises dietary fatty acids, saliva fatty acids, and those of biosynthetic origin. The saliva flow has to be known: in ovines it is 6-10 li­ ters and in bovines 50-80 liters in 24 hours (Annison and Lewis, 1959, p. 17). Furthermore, the daily average flow of ruminal content from the rumen reticulum to the intestinal tract beyond is about 7 liters in sheep and 150-170 liters in bovines (Hyden, 1961). Since it appears that no significant absorption is effected by the ruminal mucosa, nor is

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any secretion of fatty acids operative, and there is no appreciable deg­ radation in rumen with respect to fatty acids (Section VI, A), rumen is an almost ideal continuous system. In such a system we have to con­ sider that the fate of rumen microorganisms is the same as that of the liquor. The outflow from the rumen is 6 - 8 % of the contents per hour. Therefore, if a particular species of microorganisms is present in a rumen in roughly constant numbers, its mean generation time must be about 12 hours (Howard, 1967). As far as we know, this may represent the maximum rate at which protozoans can grow, but for bacteria, many of which can divide two or three times an hour, the rate of growth in the rumen must be far less than the maximum of which they are capable. The bacterial suspension in the rumen is thus a metabo­ lizing, rather than a growing, one. In the animal in vivo, not only rumen and digestive apparatus are studied, but also the effects on tissues, mainly blood and milk. In the slaughtered animals specific samples may be taken, and depot fats are those mostly considered (Section VII). The in vitro experiments are done with an artificial rumen system, in which anaerobiosis and p H values are thought to be comparable to those observed in vivo. When such conditions are maintained, precur­ sors or fatty acids may be incubated with whole rumen content, rumen fluid, mixed rumen microorganisms, washed mixed rumen microorga­ nisms, mixed rumen bacteria, mixed rumen protozoa, rumen cell-free supernatant, pure cultures of rumen bacteria, or pure cultures of rumen protozoa (Sections III and IV). 2. Lipid Extraction

and

Fractionation

In the in vivo and in vitro experiments, total fatty acids or fatty acids in different lipid fractions are analyzed. An important problem in studying rumen long-chain fatty acids not only is to obtain total fatty acids, but also to find a suitable method for extracting all natural forms of the fatty acids, directly from the fresh rumen liquor, without damage or destruction of the complexes in which they occur. The high percentage of free fatty acids (FFA), and the appreciable quantities of bacterial lipids in rumen liquor, have been a cause of difficulty. Viviani et al. (1966) have demonstrated that long-chain fatty acids of the rumen liquor of sheep are present in three major fractions, distin­ guishable also by their fatty acid spectrum; free fatty acids (FFA), esterified fatty acids, and bound fatty acids.

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At the quoted values of p H for rumen content (5-7.5), FFA could be in anionic form and therefore might be bound to cationic sites of rumen liquor proteins or of organic compounds, as occurs in vivo in plasma proteins and in animal tissues (Dole, 1956; Dole and Meinertz, 1960) and in vitro in purified serum albumins and lipoproteins (Goodman, 1958). The FFA could thus escape simple solvent extrac­ tion, and complete extraction would be obtained only after adequate acidification of rumen liquor, as is necessary for FFA of plasma pro­ teins and animal tissues. The amount of FFA extracted is d e p e n d e n t on the p H of the rumen liquor. The maximum amount of FFA is obtained a t p H 2.0. This indi­ cates that extraction without an accurate study of p H will fail to re­ cover a considerable portion of fatty acids for the subsequent proce­ dure of separation of lipid classes. Of the total fatty acids, free fatty acids range from 40 to 8 0 % (Viviani et al, 1966; Felinsky et al, 1964), depending, probably, on time after food intake. Lipids can be extracted from rumen bacteria and protozoa by the classic procedure of Folch et al. (1957). Subsequent separation of neu­ tral fats from polar lipids can be achieved, and polar lipids may be fur­ ther separated into their various fractions by column chromatography on alumina (Viviani et al., 1968a), or silicic acid (Keeney et al., 1962a), or DEAE-cellulose (Katz and Keeney, 1967). Free fatty acids can be also separated from more complex components of the nonpolar lipid fraction (Keeney et al., 1962a). 3. Fatty Acid

Determination

Lipids extracted from the material to be analyzed are complex mix­ tures; after separation of the various classes, the problem arises of the determination and isolation of the constituent fatty acids. The most commonly used methods are combinations of column and thin-layer chromatography (TLC) with gas-liquid chromatography (GLC). In the case of unsaturated fatty acids of the rumen, various methods have b e e n used, such as formation of mercury adducts (Hansen, 1966), or chromatography on silicic acid impregnated with silver nitrate (Vi­ viani and Borgatti, 1967a). For separation of multibranched acids, Hansen (1966) first separated unsaturated acids and then studied the saturated acid fraction. Urea inclusion complexes may be used for investigations of multibranched fatty acids (Ackman and Hooper, 1968). A more detailed analysis and structural determination of fatty acids may be done by a number of physical and chemical methods. Different types of spectrography — ultraviolet (UV), infrared (IR),

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nuclear magnetic resonance, and mass spectrography — may be used in association with oxidation and degradation and subsequent gaschromatographic analysis of the fragments. The methods most fre­ quently used have been UV spectrography for demonstration of conju­ gated double bonds and infrared spectrography for cis and trans isomers (Section IV, C). II.

Naturally Occurring Fatty Acids in Actual and Potential Feeds

Alimentary lipids of the ruminant have different origins. Besides lipids contained in forages, domestic ruminants are fed fermented products as silages, in which microorganisms are present, and concen­ trates, in which are present not only vegetable products but also prod­ ucts of animal origin; furthermore large amounts of animal or vege­ table materials deriving from industrial processes can be used as food. Wild ruminants can eat vegetables other than the common forages; some domestic ruminants, such as reindeer, eat mosses and lichens. Algal products can also be used as food. We think it useful, therefore, to recall the main views on fatty acid content of microorganisms, of cormophyta, and of animals. Fatty acids occur predominantly in esterified form, as glycerides or as phospholipids or other polar lipids. The fatty acids normally occurring in vege­ table and animal organisms vary in chain length from 2 to 24 carbon atoms. They may be simple straight chain, saturated or unsaturated fatty acids, acids branched at various positions, or cyclic fatty acids (Shorland, 1962; Hilditch, 1956; Hilditch and Williams, 1964). A.

MICROORGANISMS

Fatty acid composition of bacteria is reported in reviews by O'Leary (1962) and Kates (1964), that of algae and fungi by Shaw (1966), and that of protozoans by Shaw (1966) and Dewey (1967). Here we empha­ size the most important aspects according to these authors. In Enterobacteriaceae there are high levels of cyclopropane acids and low levels of C 16:1 and C 18:1 during the stationary phase of growth; during the active growth phase or at low temperature, there is an in­ version of the proportions between cyclopropane and monoenoic fatty acids, and hydroxy acids are also found. In Azobacteriaceae there are high levels of palmitic, C 16:1, cis-vaccenic acids, and of hydroxy acids, and there are no cyclopropane acids. In Lactobacillaceae also there are lactobacillic, palmitic, and cis-vaccenic acids, but C 17 -cyclopropane acid is absent. In Pseudomonadaceae, in addition to C 16:0,

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C 18:1, and cis-vaccenic acid, C 18:2 has b e e n found (Romero and Brenner, 1966); Bacillaceae have large quantities of C 15:0 and C 17:0 branched acids and low levels of the usual saturated and unsaturated acids and also fatty aldehydes (Goldfine, 1964). All members of Micrococcaceae have high proportions of branched anteiso acids. Corynebacteriaceae have high levels of branched or hydroxylated fatty acids. Complex mycolic acids of high molecular weight (C83) are found in Mycobacteriaceae. In these families also 10-methylstearic acid is found together with small amounts of the regular branched acids. Acids of the mycolic type, 10-methylstearic acid, and branched acids are also typical of Actinomycetaceae. The gram-negative bacteria have high contents of lipids, whereas the walls of gram-positive bacteria have little or no lipids. On the other hand, it appears from the collected data (Lennarz, 1966) that the gram-positive bacteria (except lactobacilli) contain high levels of the branched-chain fatty acids. Little or no branched fatty acids have b e e n found in gram-negative organisms; these contain normal saturated and unsaturated acids and cyclopropane fatty acids. As for unicellular organisms of the higher protista, while in algae normal-chain acids are found, saturated and unsaturated u p to 18:3, in Euglena gracilis unsaturated acids to 22:6 are present. Of particular interest are polyunsaturated acids C 16:3 and C 16:4 in Scenedesmus, Euglena, Cholorella, and diatoms, and also C 17:3 and C 19:4 in Euglena. Some aerobic protozoans, such as Tetrahymena pyriformis, contain 18:3 (y-linolenic type). Other protozoans, such as Leishmania, contain polyunsaturated acids and also C 22:6 (Shaw, 1966; Dewey, 1967). It is known that yeasts and molds, when cultured in adequate media, can give a high percentage of fats. Fatty acids are similar to those of certain seeds with high levels of oleic and linoleic acids. Linoleic acid content may reach 4 9 % of total fatty acids. Linolenic acid is usually present in trace amounts, but in Neurospora crass a it amounts to 3 2 % of total fatty acids (Todd et al, 1957). B. CORMOPHYTA

1. Leaf Fat Lipids of Cormophyta are studied particularly in leaves, fruits, and seeds. It appears that the lipid content of the leaf fats, including espe­ cially pasture plants, does not usually exceed 7%, and most of them are polar lipids: glycolipids and phospholipids. T h e analysis of ryegrass (Lolium perenne) and of mixed pasture grasses (Table I), used to

2.9

C 12:0

0.3

C 12:1

1.4

3.3 1.1

C 14:0

0.4

0.4

C 14:1 9.4 15.9 8.9 11.2

C 16:0 3.0 2.5 7.9 6.4

C 16:1

Fatty acids

1.5 2.0 2.8 2.6

C 18:0

13-19 3.4 9.5 12-19

C 18:1

"Expressed as percentage of total. Other data on forage fatty acids are presented in Tables X and XXII. 6 Garton(1959). c Garton (1960). d S h o r l a n d ^ a / . (1955).

Clover-rich pasture** Dactylis glomeratab

Mixed pasture grasses

Kind of forage

Table I

C O M P O N E N T F A T T Y A C I D S O F L I P I D S O F VARIOUS F O R A G E S "

20-26 13.2 8.1 19-26

C 18:2

30-39 61.3 58.9 38

C 18:3

0.7 0.5 3.9 1.5

Others

ROMANO VIVIANI

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illustrate the fatty acid composition of leaf fats, indicates high levels of linolenic and linoleic acids. Other leaf fats, such as those of spinach (Spinacia oleracea), lucerne (Medicago sativa), and buckwheat (Polygonum fagopyrum), also show that linolenic acid is the main con­ stituent (Shorland, 1962; Hilditch and Williams, 1964). The fatty acid composition of the leaf fats of the Cruciferae appears to be similar to that of the Gramineae. In the rape leaf (Brassica napus), however, in addition to a high content of highly unsaturated C 18 acids, there are also substantial amounts (11-17%) of C 16 unsatu­ rated acids consisting mainly of hexadeca-7,10,13-trienoic acid, which appears to be characteristic of rape leaf (Shorland, 1962). The most substantial deviation in the fatty acid composition of leaf material is to be found in nettle (Urtica dioica) leaf fat, which has mainly oleic acid, 82-86%, with 13-14% linoleic acid, 0.5% saturated acids, and perhaps some linolenic acid. Leaf fats are predominantly rich in linolenic and linoleic (or isomers) acids, with oleic acid usually a minor constituent. Recent investigations indicate that, in phosphatidylglycerol of chloroplasts of Leguminosae and Gramineae, in addition to a-linoleic acid, hexadecaenoic A3-trans acid (Weenink and Shorland, 1964), is present. Bark, stems, and roots have not been studied extensively, neverthe­ less it is known that in roots of the mangel (Beta rapa vulgaris) there is present erucic acid (docos-cis-13-enoic acid). In contrast to the accepted view [reported in reviews by Hilditch and Williams (1964), Shorland (1962), and Deuel (1957)], that arachidonic acid is peculiar to animals and unicellular photosynthetic orga­ nisms, Gellerman and Schlenk (1963, 1964) demonstrated in mosses and in ferns the presence of 20:4 A5-8-11-14 and also 20:5 A5-8-11-14-17. Nevertheless, we can say that in phanerogams there are no polyunsaturated acids above 18:3, if we except Ginkgo biloba in whose leaves is found a 20:4 A5-11-14-17 (Schlenk and Gellerman, 1965). It is, however, a fatty acid having a tetramethylenic interruption between the first and second double bond. 2. Fruit-Coat

Fats

The fleshy or succulent parts of fruits contain, at times, considerable proportions of neutral fat. Olea europaea (olive oil), Lauris nobilis (laurel oil), and Elaeis guineensis (palm oil) contain palmitic, oleic, and linoleic acid; in laurel oil, a high quantity of lauric acid is present; Stillingia sebifera (stillingia tallow) and Rhus sp. (sumach tallow) con­ tain higher levels of palmitic and oleic acid, and linoleic acid is absent (Shorland, 1962).

C 16:1 (t3), C 16:1 (t5) C 18:1 (t3), C 18:2(c9,tl2) C 18:3 (t3,c9,cl2), C 18:3 (t5,c9,cl2) Vernolic acid: 12-13 epoxy-cis-9octadecenoic acid a-Eleostearic acid: C 18:3 (c9,tll,tl3) Licanic acid: 4-keto-octadeca-cis-9, frans-11, trans-13-trienoic acid Parinaric acid: octadeca-9,11,13,15tetraenoic acid a-Eleostearic acid: C 18:3 (c9, tll,tl3) Ricinoleic acid: 12-hydroxy-cis-9-octadecenoic acid co-Hydroxyeleostearic acid Punicic acid: C 18:3 (c9,tll,cl3)

C 18:4 (c6,c9,cl2,cl5) 6

Characteristic fatty acids

Cucurbitaceae

Euphorbiaceae

Rosaceae

Compositae

Coniferae, Moraceae, Celastraceae, Aquifoliaceae, Labiatae, Valerianaceae, Onagraceae, Linaceae, Elaeagnaceae, Rhamnaceae, Nyctaginaceae, Dilleniaceae, Ericaceae, Boraginaceae, Juglandaceae, Hippocastanaceae, Ulmaceae, Olacaceae, Vitaceae, Papaveraceae, Betulaceae, Passifloraceae, Typhaceae, Theaceae, Fugaceae, Solanaceae, Pedaliaceae, Scrophulariaceae, Plantaginaceae, Calycanthaceae, Campanulaceae, Symplocaceae, Polemoniaceae, Chenopodiaceae, Hamamelidaceae, Loasaceae, Portulacaceae, Agavaceae, Liliaceae, Proteaceae, Asclepiadaceae, Staphyleaceae, Dispacaceae, Myrtaceae Urticaceae, Oleaceae, Ranunculaceae, Compositae

Botanical families

M A J O R AND C H A R A C T E R I S T I C F A T T Y A C I D S IN S E E D F A T S O F T H E BOTANICAL F A M I L I E S "

Linoleic, oleic, linolenic, and conjugated polytenoid acids

Linoleic and linolenic (oleic) acids

Linolenic and linoleic (oleic) acids

Major fatty acids

Table II:

to

2 O

00

ROMANO VIVIANI

T h e data collected in this table are derived from the following references: Shorland (1962); Hilditch and Williams (1964); Dinh-Nguyen (1967). "That is, octadeca-cis-6,cis-9,cis-12,cis-15-tetraenoic acid; c = cisy t = trans.

Capric acid

Sterculiaceae Sapindaceae, Buxaceae Cruciferae Oleaceae Leguminosae, Moringaceae, Ochmaceae, Sapindaceae Gnetaceae, Meliaceae, Menisphermaceae, Sterculiaceae, Guttiferae, Dipterocarpaceae, Bursuraceae, Sapotaceae, Convolvulaceae, Verbenaceae Lauraceae, Myristicaceae, Simarubaceae, Vochysiaceae, Salvadoraceae, Palmae Ulmaceae

Flacourtiaceae

Umbelliferae, Araliaceae Simarubaceae Olacaceae, Santalaceae

CD

to

Fatty Acids in the Rumen

a

Myristic and lauric acids

9-Hydroxyoctadec-12-enoic acid Sterculic acid (cyclopropenic acid) C 18:2 (U0,tl2) C 18:2 (c9,tl2) C 18:3 (t9,tll,cl3) C 18:4 (t3,c9,cl2,cl5) Petroselinic acid: C 18:1 (c6) Tariric (octadec-6-ynoic) Ximenynic acid (octadec-ll-en-9-ynoic acid) Cyclic unsaturated acid (chaulmoogric acid) Sterculic acid (cyclopropenic acid) C 2 0 : l (ell) Erucicacid: C 22:1 (cl3) Higher than erucic acid Lignoceric, and cerotic acids Stearic acid

Gramineae, Berberidaceae, Magnoliaceae, Anonaceae, Rutaceae, Zygophyllaceae, Anacardiaceae, Capparidaceae, Hernandiaceae, Tiliaceae, Malvaceae, Bombacaceae, Caryocaraceae, Caricaceae, Lecythidaceae, Combretaceae, Apocynaceae, Amaranthaceae, Martyniaceae, Acanthaceae, Bignoniaceae, Rubiaceae, Caprifoliaceae Apocyanaceae Malvaceae Bignoniaceae

of Long-Chain

Characteristic acids other than, or in addition to, oleic, linoleic, and palmitic acids

Palmitic, oleic, and linoleic acids

Metabolism

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3. Seed Fats T h e lipids of seeds are principally neutral fat; however small quan­ tities of polar lipids are present. In wheat germ, for example, neutral fats represent 9 7 % of total lipids (Moruzzi et ah, 1969). The classification of the seed fats on the basis of their chief compo­ nent fatty acids, as outlined by Hilditch and Williams (1964), is fol­ lowed in this subsection. Since the data regarding seed fats are volu­ minous, we list in Table II the most significant data; in some cases the same family may appear under two different classifications for better indication of specific fatty acids. Seeds utilized in practical diets of ruminants are mostly of Gramineae and Leguminosae, but there are seeds of other families that have actual and potential importance; these are of various compositions, with particular fatty acids, the pres­ ence of which must always be considered (Table II). C.

ANIMALS

The fatty acid composition of animals (Metazoa) is reported by Hil­ ditch and Williams (1964). Comparative considerations are referred by Shorland(1962). Studies of fatty acid composition of insects have not received as much attention as studies of acids of other animals or of vegetables. Saha et al. (1966) found no polyunsaturated acids other than C 18:3 in insects. In marine invertebrates there are polyunsaturated acids; a holothurian found a depth of 4400 meters contained arachidonic acid (Lewis, 1967). As for polyunsaturated acids, terrestrial animals have more arachidonic acid, whereas marine animals contain high levels of C 20:5, and C 22:6 (Hilditch and Williams, 1964; Shorland, 1962; Lovern, 1964). III. A.

Fatty Acid Composition of Rumen Microorganisms

BACTERIA

1. Mixed Rumen

Bacteria

Lipids of washed mixed ovine rumen bacteria are about 7 0 % nonphospholipids and 3 0 % phospholipids (Table III). It is to be noted (Viviani et al., 1968a) (Table III) that in rumen bac­ teria lecithin content is extremely low, but the phosphatidylethanolamine and phosphatidylserine fractions are present in high amounts — a general characteristic of bacteria (Law, 1967).

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Fatty Acids in the Rumen

281

Table I I I L I P I D C O M P O S I T I O N O F M I X E D R U M E N B A C T E R I A AND P R O T O Z O A 0

Lipid Phospholipids Nonphospholipid Free fatty acids Phosphatidylcholine Phosphatidylcholine plasmalogen Phosphatidylethanolamine Phosphatidylethanolamine plasmalogen Aminoethylphosphonate plasmalogen Phosphatidylserine Phosphatidylinositol Phosphatidic acid Diglyceride aminoethylphosphonate Glyceryl ether phospholipids Unidentified: containing neutral amino acid Ceramide aminoethylphosphonate Ceramide ethanolamine phosphate Sphingomyelin Unidentified

Protozoa

Bacteria b

b

c

d

e

30.05 69.95 40.39 1.20

27.35 72.65 63.68 30.71

70 30 9 28

— -

— -

-

-

-

66.59

54.86

-



21.59

2.80

— — -

— -

11.81

11.49

21 22

— -

29

36.30 0.90 18.70 9.50 1.90 0 3.10 1.40 11.00 4.50 3.60 4.10 2.20 2.00 0.80

54

— — -

°The data for phospholipid, nonphospholipid, and free fatty acids are expressed as percent of total lipids; all other data, as percent of lipid P. b W\w\QXii etal. (1968a). c Katz and Keeney (1967). d Dawson and Kemp (1967). e Gutierrez e* aZ. (1963).

Fatty acid composition of mixed rumen bacteria has b e e n investi­ gated both in bovines and in ovines. In mixed bacteria of ovine ru­ men, fatty acids have b e e n studied in total lipids (Hansen, 1966; Vi­ viani et al., 1968a) (Table IV), in neutral fat, and in phospholipids (Viviani et al, 1963a, 1968a; Keeney et al, 1962a) (Table V). The dietary fatty acids (Table I) differ from those of ruminal bacteria not only in linoleic and linolenic acids, but also in the presence of odd or branched acids, which are not present in dietary lipids. T h e pres­ ence of C 18:2 in higher amounts in phospholipids shows a real utili­ zation of this acid for protoplasmic constituents of bacteria. The pres­ ence of polyunsaturated acids was not previously observed (Garton and Oxford, 1955). T h e fatty acid distribution in various polar lipid classes is also re­ ported (Viviani et al, 1968a). T h e typical fatty acids of rumen bacteria (branched and odd acids) are found mainly in the phosphatidyl­ ethanolamine fraction (Table VI).

282

ROMANO VIVIANI Table IV TOTAL FATTY ACID COMPOSITION (WEIGHT PERCENT) O F OVINE RUMEN R A C T E R I A AND P R O T O Z O A

Mixed rumen bacteria Fatty acids C 12:0 C 13:0 C 13:0 C 14:0 C 14:0 C 15:0 C 15:0 C 16:0 C 16:0 C 16:1 C 17:0 C 20:0 C 17:0 C 18:0 C 18:0 C 18:1 C 19:0 C 18:2 C20:0 C 18:3 Others

br c br br br

br br br

a

b

L15 0.61 0.82 1.23 3.94 12.73 8.00 1.15 30.95 4.02

07 0.7 1.1 1.7 4.1 16.0 8.4 1.1 30.0

— —

3.3 2.8 1.8 0.1 6.9 11.3 Traces 4.10 0.50 1.20 4.20

1.56 0.13 14.95 5.99 0.09 2.71 0.10 1.03 8.74



Mixed rumen protozoa 0 0.21 0.19 0.46 0.48 1.48 4.25 4.28 2.26 37.83 6.78

— —

1.37 0.15 13.48 11.48 0.04 6.34 0.10 4.74 4.19

a

Viviani e* aZ. (1968a) Hansen (1966). c br = branched. 6

T h e time after the intake of food affects the fatty acid composition of mixed rumen bacteria (Viviani et al., 1963a). In this respect, it has b e e n observed that linoleic acid decreases after 24 hours in accord­ ance with the hydrogenating activity of bacteria. Investigations by Keeney and Katz (1966) in relation to time after feeding (from 0 to 4 hours) did not reveal any changes either of total lipids or of fatty acids, or of the concentration of frans-octadecenoic acid. According to Keeney and Katz (1966), whereas octadecenoic acid from rumen digesta consisted of 7 5 % trans acid, with A11 as the major acid, in rumen bacteria octadecenoic acids consisted of a mixture of 24 geometrical and positional isomers. The major acids are octadecenoic acids (trans-ll;cis-9;cis-ll). T h e analysis of bacterial lipids indicates that the rumen bacteria preferentially esterify the cis-isomers into polar lipids.

br

br

br

br

br

Keeney et al. (1962a). "Viviani et al. (1968a).

a

0.6 Traces Traces 0.7 2.0 6.7 4.5 2.9 26.1 1.5 1.5 0.4

C 12:0 C 13:0 C13:0 C 14:0 C 14:0 C 15:0 C 15:0 C 16:0 C 16:0 C 16:1 C 17:0 C 17:0 C 18:0 C 18:0 C 18:1 C18:2 C 18:3 Others

7.9



10.7 16.2 18.1

1.4 1.4 21.1 6.8 3.0 1.0 6.0

0.5 0.3 0.5 1.0 3.3 10.9 6.4 1.2 31.4 4.1

Ovine

— -

6.5 10.2 10.7



L5 1.2 0.7 1.3 3.8 20.3 8.2 1.6 30.6 1.3 1.2 0.8

Bovine

Polar lipids

1.5 0.2 7.4 8.1 5.9 1.2 9.5

— -

58.9 12.5 4.3

-

16.9 0.8 0.5 1.9

1.2 0.1 22.8 6.4 2.0 1.1 8.8

2.3 0.5 0.8 1.1 4.0 9.8 7.1 0.2 29.7 2.2

Traces Traces Traces Traces 0.9 1.9 1.2

0.1 Traces 0.1 0.7 2.7 11.7 8.8 1.8 34.3 5.4



Ovine

Free fatty acids Bovine

Ovine

of Long-Chain



Bovine

Neutral lipids

Fatty acids

br

Table V

F A T T Y A C I D C O M P O S I T I O N ( W E I G H T P E R C E N T ) O F L I P I D S IN B O V I N E 0 AND O V I N E " R U M E N B A C T E R I A

Metabolism GO

to

00

Fatty Acids in the Rumen

284

ROMANO VIVIANI Table VI

FATTY ACID COMPOSITION OF LIPID POLAR FRACTIONS O F RUMEN

Fractions

Fatty acid C 12:0 C 12:1 C 13:0 C 13:1 C 14:0 C 14:1 C 15:0 C 15:1 C 16:0 C 16:1 C 17:0 C 18:0 C 18:0 C 18:1 C 19:0 C 18:2 C20:0 C 18:3 Others

plus 13:0 br plus 14:0 br plus 15:0 br plus 16:0 br plus 17:0 br br

Unknown

oil

0.05 0.27 0.99 3.02 7.17 10.36 2.35 35.48 6.62 1.48 0.20 9.42 10.85



4.59 0.21 1.59 5.11

BACTERIA"

6

Containing phosphatidylethanolamine

Containing phosphatidylserine

0.54 0.44 0.69 1.56 4.08 17.83 10.56 1.87 30.50 5.23 1.54 0.16 7.40 6.97

Traces Traces Traces 0.59 2.41 13.73 5.96 0.87 28.91 3.13 1.77 0.55 12.50 10.49 0.17 4.31 0.41 1.18 13.23



3.41

— 1.15 6.08

a

Vivianie*aZ. (1968a). T r a c t i o n s were obtained by column chromatography on alumina (Webster, 1960); phospholipid components in each fraction were checked by chromatography on silicic acid-impregnated paper (Marinetti, 1964). Values are expressed as weight percent cal­ culated from peak areas.

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), which hitherto has been isolated and identified from several natural sources including butterfat, ox periphrenic fat, and cow plasma, has been found by Hansen (1966), in small amount (2.9%) in the total fatty acids extracted from the rumen bacteria of a dairy cow fed a diet of clovergrass-hay. This C 20 multibranched fatty acid was not detected in dietary clover-grass; it is considered to have b e e n derived from phytol by enzyme processes of the rumen bacteria. 2. Cellulose Digesting

Bacteria

T h e most typical rumen bacteria are those that digest cellulose; they are either rod shaped or cocci. The main cellulolytic bacteria are Ruminococcus flavefaciens and

Metabolism

of Long-Chain

Fatty Acids in the Rumen

285

Ruminococcus alhus; an analysis of the fatty acids of a pure culture of Ruminococcus flavefaciens has b e e n undertaken by Allison et al. (1962), who found that this organism contains about 2 4 % of C 15 branched-chain acids (Table VII). 3. Starch Digesting

Bacteria

T h e noncellulolytic species Streptococcus bovis, Bacteroides amylophilus, Bacteroides ruminicola, and Selenomonas ruminantium in­ clude many starch digesters; it is known that w h e n alfalfa hay-fed animals are given excess grain, the number of S. bovis increases al­ most explosively (Hungate et al., 1952; Krogh, 1959) and can cause fatal indigestion due to the high concentration of lactic acid produced. It is to be noted that the highest levels of S. ruminantium have b e e n observed (20-30% of the total colony count) in steers fed cracked corn and urea. T h e fatty acid composition of two strains of Streptococcus bovis and several of Selenomonas ruminantium has b e e n studied (Table VII). Streptococcus bovis is characterized by a rather simple spectrum, with high amounts of C 16:0 (28-21%), of C 16:1 (16-9%), of stearic acid (15-10%), and of C 18:1 (20-24%). It is therefore pos­ sible that this strain is active for the synthesis of vaccenic acid, without excluding the possibility of an accumulation of C 18:1 from the cultural medium (Viviani et al., 1967b). The strains of Selenomonas ruminantium all contain high levels of odd straight-chain fatty acids; only in few strains are detectable quan­ tities of branched-chain fatty acids found, comprising up to 1 5 % of the total fatty acids (Table VII). Other investigations (Viviani et al., 1967b) have shown that 5-day cultures are all constituted of vibrionic forms whereas 10-day cultures have cells of round degenerative shape. The fatty acid composition of these strains having different morphology did not show variations. Shape modifications in S. ruminantium do not appear to be accompanied by important alterations of the most characteristic lipid structures. These data indicate that starch-fermenting bacteria have high levels of normal saturated or unsaturated or odd fatty acids, while branched fatty acids are scarce. 4. Bacteria Digesting

Hemicellulose

and Pectic

Substances

The ability to digest hemicellulose is characteristic of all cellulolytic strains. Also Succinivibrio dextrinosolvens, xylan fermenter typical of rumen, contains high levels of branched acids (Viviani et al, 1967b) (Table VII). Pectin, too, is rapidly fermented in the rumen. None of the typical

286

ROMANO VIVIANI

Table VII FATTY ACID COMPOSITION O F SOME TYPICAL

Microorganism

C10:0

0.08 Selenomonas ruminantium 6a& Selenomonas ruminantium 105 cb — 0.12 Selenomonas ruminantium 105 d 6 0.71 Selenomonas ruminantium 105 e 6 0.24 Selenomonas ruminantium 105 fb Selenomonas ruminantium 105 l6 Traces 0.15 Selenomonas ruminantium 106 eb 1.17 Selenomonas ruminantium 106 fb Succinivibrio dextrinosolvens 5 ab — 0.20 Butyrivibrio sp. 14 ax6 0.01 Butyrivibrio sp. 12 ax6 Streptococcus bovis 18/C2 6 — 0.19 Streptococcus bovis 2 SAft 0.12 Peptostreptococcus elsdeniib Buminococcus albusc — Buminococcus flavefaciens C 94 c — d Bacillus polymyxa 8517 -

Microorganism Selenomonas ruminantium 6a 6 Selenomonas ruminantium 105 cb Selenomonas ruminantium 105 d 6 Selenomonas ruminantium 105 eb Selenomonas ruminantium 105 fb Selenomonas ruminantium 105 P Selenomonas ruminantium 106 eb Selenomonas ruminantium 106 fb Succinivibrio dextrinosolvens 5 a6 Butyrivibrio sp. 14 ax6 Butyrivibrio sp. 12 axft Streptococcus bovis 18/C2ft Streptococcus bovis 2 SAft Peptostreptococcus elsdeniib Buminococcus albusc Buminococcus flavefaciens C 94 c Bacillus polymyxa 8517d

C17:0 br 0.50 0.21 3.07 Traces 1.01 Traces



Traces 1.58 1.42 2.05

-

28.83

C11:0 br

_ 0.15 0.65 3.57 2.25 0.34 1.19 3.26



0.05

— — — — -

C11:0

C12:0 br



0.28 0.73 1.58 5.09 3.04 2.07 2.39 3.66 0.01

0.29 0.23 1.01 2.03 0.56 1.49 2.66

-

0.09

— -

— -

— — -

C17:0

C17:l

C18:0 br

4.86 1.62 2.33 2.68 2.00 3.25 1.43 1.58 2.49 1.62 1.91 1.79 4.29 6.08 0.80

1.41 0.25 0.61 0.83 0.52 1.80 1.52 1.77



0.08 1.16

0.23 1.25

2.91 1.30 0.43 0.62 0.90 0.90 0.33 0.79 1.04 1.82 2.41 0.47 0.21 3.31

— —

— —

— — -

C12:0 0.68 3.97 6.24 8.32 9.47 6.76 7.99 6.41 0.85 0.25 0.73 0.20 1.34 6.15

— -

C12:l

RUMEN

C13:0 br

— Traces 1.19 1.95 — — — 1.39 — 3.56 0.27 0.13 0.42 0.07 — 0.21

-

0.10 1.20

-

0.20

— — -

C18:0

C18:l C19:0

11.66 9.29 6.64 6.88 6.76 7.94 11.64 6.53 15.13 13.59 6.92 15.13 10.49 8.54 1.20 9.20 1.10

5.81 6.15 5.16 2.99 4.02 4.14 6.93 3.49 4.81 8.78 2.13 20.51 24.33 19.77

11.53 1.39 2.70 3.32 3.91 1.80 3.93 3.60 0.56 1.37 0.35 0.97 1.91 4.39

— -

— —

1.47

287

Metabolism of Long-Chain Fatty Acids in the Rumen

BACTERIA (PERCENT OF TOTAL FATTY ACIDS) C13:0 1.60 2.41 1.52 5.13 4.20 3.12 3.04 6.17 0.18 0.04 0.18 1.16

C13:1 0.17 0.52 Traces Traces Traces Traces 0.20 Traces 0.16 1.01 Traces

1.00 1.77

C18:2 2.29 1.65 0.34 0.66 Traces 0.74 2.82 0.64 0.21 3.38 0.13 0.39 0.20 0.50

C20:0 br

1.50 0.76 0.01 0.20 2.05

C14:0 br

C14:0

C14:1

C15:0 br

2.36 5.75 5.07 5.93 6.60 6.15 6.84 5.07 6.37 7.37 12.47 3.82 6.31 3.83 3.10 4.90 1.90

2.75 4.21 2.60 3.02 2.91 2.90 3.01 4.81 0.30 2.01 0.61 0.89 1.11 2.21

0.71 0.93 6.09 1.22 0.44 1.54 0.55 0.91 17.26 2.39 5.80 2.65 2.402.37

0.16 0.91 0.86 3.21 1.18 0.77 1.01 3.96 1.29 0.77 0.96 0.05 0.13 0.36 38.80

C15:0

C15:1

8.95 8.31 4.59 8.41 6.27 10.19 5.59 7.06 6.34 2.91 4.88 2.05 5.74 8.84 1.60 2.10

8.54 3.00 2.06 2.36 2.35 6.00 3.01 3.02

24.60 40.28

C20:0

C20:1

Xla

X2

C22:0

7.34 3.66 3.81 3.04 7.97 3.32 2.26 2.30 0.64 0.86 0.37 0.60 1.96 0.67

Traces 2.62 0.78 0.93 1.12 0.75 4.92 0.36

1.84 3.84 5.93 4.29 3.04 2.95 4.67 3.27

2.60

1.27 1.68 1.93 1.10 0.93 0.82

0.42 2.74 0.92 0.91

0.73

aXl, X2, X3 = unidentified. bYiviani et al. (1967b).

cAllison et al. (1962). dYiviani et al. (1965).

0.30

C16:0 br

C16:0

C16:1

0.95 0.90 0.90 0.71 1.20 0.81 0.31 0.79 5.19 3.47 3.30 0.50 Traces 0.96 12.80 5.60 7.20

10.00 14.69 10.80 9.53 10.44 12.02 10.87 9.76 27.77 36.05 44.51 28.32 21.35 17.25 38.60 20.60 16.73

8.78 8.11 8.50 2.67 10.17 11.94 3.14 3.22 4.03 9.14 9.23 16.00 9.20 6.41

X3

C23:0

3.45 2.49 6.42 3.22 3.33 4.52 5.47

5.26 9.05 5.00 2.81 3.09 3.48 3.81

Unidentified aboveC 21:0

2.39 1.48 0.27 2.74 5.85 1.98

288

ROMANO VIVIANI

pectinolytic soil bacteria has been found in rumen, if we except Bacillus polymyxa (Zambonelli and Matteuzzi, 1964). Fatty acid compo­ sition of B. polymyxa isolated from ovine rumen is completely similar to that of B. polymyxa ATCC 10401, producer of polymyxin, and con­ tains high levels of iso and anteiso branched acids, approaching 7 0 % of total fatty acids (Viviani et al, 1965) (Table VII). 5. Sugar Fermenting

Bacteria

All the polysaccharide-digesting bacteria can also utilize mono- or disaccharides, although lactobacilli also are typical sugar fermenters. Several species of lactobacilli appear in the rumen (Hungate, 1966, p. 75). They are an important component of the rumen flora of young calves and on occasion are found in large numbers in the adult rumen. Determinations of fatty acids in ruminal lactobacilli are lacking. A fact to be noted is that lactobacillic acid has not b e e n observed in rumen (Allison et a/., 1962; Felinsky et al., 1964; Viviani et al, 1963a; Hansen, 1966), if we except a reference by Reiser et al., (1963). More­ over, this acid has not been found in milk (Jensen et ah, 1967). This might indicate usually the low amount of lactobacilli in rumen, or also that ecologic conditions of rumen are not proper for the production of the cyclopropane ring. 6. Bacteria Utilizing

Acids

In addition to Selenomonas ruminantium, which utilizes lactate or propionate, many rumen bacteria can decompose lactate, succinate, or formate. Among lactate-utilizing bacteria, are Veillonella gazogenes and Bepto streptococcus elsdenii. These organisms may be important fermenters of lactate in cattle during adaptation to a high grain ration conducive to feedlot bloat (Gutierrez et al., 1959). They occur in con­ junction with Streptococcus bovis and other bacteria that ferment starch to lactate and are abundant in the rumen of young calves but subsequently decrease. P. elsdenii contains, in addition to normal, even saturated (36%) and unsaturated (28%), also odd saturated (21%) and branched (9%) acids. Although data on the fatty acid content in lipolytic bacteria and in methanogenic and in proteolytic bacteria are lacking, the data now available on typical bacteria of rumen allow us to consider that branched fatty acids are generally linked to gram-positive bacteria (Lennarz, 1966; Kates, 1964), and that they are particularly abundant in cellulosolytic bacteria and in digesters of hemicellulose and pectic substances, whereas odd fatty acids are high in starch digesters and also in bacteria that utilize acids.

Metabolism

of Long-Chain

Fatty Acids in the Rumen

289

These observations show that branched- and odd-chain fatty acids found in milk and depot fats of ruminants have their origin from fatty acids of structural lipids of bacteria. B.

FUNGI

Only the yeast Candida is found in the rumen (Clarke and Di Menna, 1961). Nonetheless the importance of fungi seems to be negli­ gible. The fatty acid content of Candida sp. has b e e n studied in inves­ tigations unrelated to rumen. Besides the normal C 16:0, and C 16:1, there are oleic, linoleic, and linolenic acids (Kates and Baxter, 1962). C.

PROTOZOA

An immense population of various specialized kinds of ciliate proto­ zoans is found in the rumen of domestic ruminants. Their number according to Hungate (1966, p. 92) may exceed one million per gram of rumen content, and their biomass roughly equals that of bacteria. When we consider rumen protozoans, we usually mean ciliates, since they represent the highest biomass, but a few species of small flagellates are also regularly encountered. The flagellates are not numerous in the adult ruminant, counts of 2 X 103 to 3 X 104 being reported in sheep (Warner, 1962); because of their small size they do not compose a very large fraction of the bulk of the rumen protozoa. Various species of flagellates in rumen have been found by Jensen and Hammond (1964). All domesticated ruminants, and almost all the wild ruminants whose rumen faunas have been examined, contain at least one species of Protozoa, usually several. 1.

Rumen

Ciliates

In the rumen 100 or so ciliate species have b e e n observed. The cil­ iates of rumen fall into the family of Isotrichidae (order Trichostomatida, subclass Holotrichia), commonly referred to as the "rumen holotrichs," and into the family of Ophryoscolecidae (order Entodiniomorpha, subclass Spirotrichia) referred to as "oligotrichs" or "entodiniomorphs" (Corliss, 1967). Number and species vary de­ pending on a variety of factors, chief of which is the diet of the host. The rumen ciliates are strictly anaerobic and thrive best at an E/, be­ tween —200 and —260 mV. In Tylopoda, such as the camel, they ap­ pear to be different from those in domestic ruminants. Williams (1963) found no protozoans in Australian camels. In ruminantlike animals,

290

ROMANO VIVIANI

such as hippopotamus, (Thurston, 1963), protozoans are found similar to those of rumen. Investigations by our group (Viviani et al., 1968a) have demon­ strated that protozoans in sheep fed hay contain 3 0 % phospholipids and 7 0 % nonphospholipids. However, Katz and Keeney (1967) found that 7 0 % of lipids were phospholipids (Table III). The total lipid con­ tent of rumen ciliates was found to be 7.7%, of which 5 4 % was free fatty acids (Gutierrez et al., 1963). Fatty acid composition of sheep mixed rumen protozoans and com­ parison of fatty acids of neutral fat and polar lipids of bovine (Keeney et al., 1962a), and ovine (Viviani et al., 1968a) protozoans are reported in Tables IV and VIII. Particularly interesting are the levels of C 18:2 and C 18:3, which are higher than in bacteria. Investigations by Viviani et al. (1963a) in relation to time after feeding (from 3 to 24 hours) indicate also the presence at 24 hours, both in neutral fat and in phospholipids, of C 18:3 of possible biosynthetic origin. The fatty acid composition of various polar lipid fractions have been studied by Katz and Keeney (1967) and Viviani et al. (1968a). In Table IX are reported data from our laboratory. Particularly interesting is the low level of stearic acid in the fatty acid spectrum; this brings into question the actual extent of biohydrogenation by protozoans. Pure cultures of rumen protozoans have b e e n studied by Dawson and Kemp (1967) in relation to aminoethylphosphonate-containing phos­ pholipids. 2. Rumen

Flagellates

There are no data on lipid composition of rumen flagellates. From data in the literature, flagellates contain polyunsaturated acids (Shaw, 1966; Dewey, 1967); however, it is not possible to predict what is happening in the rumen in anaerobiosis. IV.

Fatty Acid Metabolism of Rumen Microorganisms

A. L I P I D H Y D R O L Y S I S AND A B S O R P T I O N O R A D S O R P T I O N O F FATTY ACIDS

Fatty acids in lipids of rumen bacteria and protozoans may derive from dietary fatty acids with or without modifications or may be of bio­ synthetic origin. On the other hand, it was demonstrated that bacteria can discharge complex lipids into the medium (Bishop and Work, 1965). Exogenous fatty acids contained in simple and complex dietary

9.7

_ _

27.5



12.3 17.1



1.0 0.5 1.1 2.2 26.5 0.7 1.2 Traces

— _ — _

Bovine '

a d

5.15 1.21 0.12 18.09 13.50 0.05 8.33 0.09 4.07 4.56



0.13 0.22 0.15 0.22 0.99 3.34 3.56 1.38 34.83

Ovine

Neutral lipids

b

Keeney etal. (1962a). V i v i a n i ^ a / . (1968a). c Katz and Keeney (1967). ^Protozoa are contaminated by food residue.

a

br

br

br

br

br

br

Fatty acids

C 12:0 C 13:0 C 13:0 C 14:0 C 14:0 C 15:0 C 15:0 C 16:0 C 16:0 C 16:1 C 17:0 C 17:0 C 18:0 C 18:0 C 18:1 C 19:0 C 18:2 C20:0 C 18:3 Others

Table VIII

6

3.2

— —

14.6

-



10.3 20.3

Traces Traces 0.7 Traces 1.6 3.7 2.0 1.1 37.5 1.2 2.8 0.8

Bovine"-

d

Polar lipid:5



9.38 2.52 0.24 17.26 14.28 0.34 2.78 0.22 1.89 3.83

0.02 0.02 0.09 0.09 1.22 2.08 2.26 2.19 39.30

Ovine

6

c

-

— — -

— 4.9

— 68.3 10.0

-

-

6.0

-

10.2

3.9 2.3 Traces 8.1 5.9

-

5.19 1.34 0.09 17.66 12.36 0.03 7.59 0.07 5.25 2.10

— 0.6 1.3

0.6 Traces 14.2



Traces 1.3 3.3 6.6 2.1 50.1

— — —

Traces

~b 0T2 0.10 0.16 0.52 1.58 3.97 5.25 1.89 35.08

— — — —

Bovine"-**

Ovine

Free fatty acids

F A T T Y A C I D C O M P O S I T I O N ( W E I G H T P E R C E N T ) O F L I P I D S IN B O V I N E 0 AND OVINE 6 - 0 M I X E D R U M E N P R O T O Z O A

to

CD

Metabolism of Long-Chain Fatty Acids in the Rumen

292

ROMANO VIVIANI Table IX

FATTY ACID COMPOSITION (WEIGHT PERCENT) OF LIPID POLAR FRACTIONS OF RUMEN MIXED PROTOZOANS"

Fractions 6

Fatty acid C 12:0 C 12:1 C 13:0 C 13:1 C 14:0 C 14:1 C 15:0 C 15:1 C 16:0 C 16:1 C 17:0 C 18:0 C 18:0 C 18:1 C 19:0 C 18:2 C20:0 C 18:3 Others

Containing phosphatidylcholine 0.25

plus 13:0 br plus 14:0 br plus 15:0 br plus 16:0 br plus 17:0 br br

-

0.22 0.28 2.32 4.17 4.65 2.36 33.91 5.66 1.67 0.29 11.37 18.62 0.19 7.40 0.02 3.52 3.11

Unknown 0.30 0.09 0.71 0.45 2.60 4.84 3.80 3.80 35.31 11.07 0.65 0.27 8.80 15.38 0.20 4.38 0.16 2.61 5.19

Containing phosphatidylethanolamine

Containing phosphatidyl serine

0.21 0.08 0.14 0.43 2.13 6.46 3.65 4.55 32.46 15.61 1.64 0.09 11.28 15.45

045 0.23 0.48 0.41 3.41 2.65 2.76 3.10 24.75 8.88 1.58 0.54 13.87 22.15 0.33 3.79 1.10 1.17 8.36



2.54



1.70 1.58

"Viviani etal. (1968a). ^Fractions were obtained by column chromatography on alumina (Webster, 1960); phospholipid components in each fraction were checked by chromatography on silicic acid-impregnated paper (Marinetti, 1964). Values are expressed as weight percent cal­ culated from peak area.

ormicrobial lipids may be hydrolyzed by specific enzymes. Garton et al. (1958) observed that rumen microorganisms can hydrolyze the ester linkage between fatty acid and glycerol. Other investigations showed hydrolysis of galactoglycerides, of T w e e n 80, of sterol esters, and of methyl and ethyl esters (Garton, 1965). Phospholipids can also be hydrolyzed (Dawson, 1959). As a consequence of the activity of these lipolytic enzymes, high levels of free fatty acids are found in rumen. Spectra of free fatty acids (Table X) may indicate their origins; obviously high stearic acid expresses biohydrogenation of dietary unsaturated acids, while the presence of branched and odd acids indi­ cates a microbial origin, since these acids are not contained in the diet (Viviani et al., 1963a). Both bacteria and protozoans are involved in this process. When ethyl laurate and ethyl oleate were incubated in

Table X

Traces 5.01 5.98 14.26 38.46 7.90



21.48 1.74



0.32



4.41



2.60 25.70 21.80 20.30 13.00

— — —

16.60

— — — — —

0.36 lipid of feed — Total a b — —

"Viviani etal. (1966). ft Felinskye*aZ.(1964).

C 12:0 CFatty 13:0 acid C 14:0 br C 14:0 C 15:0 br C 15:0 C 16:0 br C 16:0 C 16:1 C 17:0 br C 17:0 C 18:0 C 18:1 C 18:2 C 18:3 Other acids 0.37 0.19a acids 2.39 2.64 3.31 2.51 0.95 21.85 2.11 Traces 1.90 22.75 11.35 11.38 4.13 5.39

Tofal

Total fatty acids a feHv

b

23.02 2.76 Traces 1.72 48.08 13.06 5.67 2.15



b

— 0.27 Esterified fatty acids 0.55 a b — 2.56 — 4.24 — 3.87 — 2.49 — 1.12 — 25.90 26.50 Traces — Traces — 2.31 — 23.94 14.90 18.90 14.85 7.70 6.40 0.97 3.80 6.80 28.80

a

Esterified fatty acids

Rumen liquor

73.10 11.10 0.70 Traces 5.30

— — —

9.80



— fatty acids— Free 0.87 a b — 0.28 — 0.79 — 0.66 — 1.60 —

a

Free fatty acids

6.91

1.54 8.50 32.73 5.36



0.71 F a H v a ^ c nf "bound1.00 lipids" a Traces 1.42 Traces 0.87 Traces 29.73 8.16

Fatty acids of "bound lipids" 0

L O N G - C H A I N F A T T Y A C I D C O M P O S I T I O N ( W E I G H T P E R C E N T O F T O T A L ) O F D I E T A R Y L I P I D S AND R U M E N L I Q U O R O F S H E E P

Metabolism of Long-Chain Fatty Acids in the Rumen

294

ROMANO VIVIANI

vitro with ruminal liquor, the greater part of the total hydrolysis was ascribed to the ciliate protozoans (Hill et al., 1960). On the other hand, rumen protozoans can exert phagocytosis of whole chloroplast parti­ cles and digest them (Wright, 1959). Specific lipolytic microorganisms are motile rods with a single polar flagellum; they were isolated from the rumen on a medium containing emulsified linseed oil. The name Anaerovibrio lipolytica is proposed for this organism (Hobson and Mann, 1961). Long-chain fatty acids do not appear to undergo, after the hydrolysis by soluble enzymes, further degradation phenomena, and they are absorbed or adsorbed by bacteria and protozoa. High levels of free fatty acids are present in lipids of bacteria and proto­ zoans (Table III). Several factors may affect hydrolytic processes; thus inhibitory effects have been demonstrated by antibiotics on lipolysis (Wright, 1961). Free fatty acids may then be absorbed; in some in­ stances it was possible to demonstrate the order of fatty acid absorp­ tion by some protozoans: Isotricha prostoma concentrated fatty acids in the following order: stearate, oleate, palmitate, linoleate; Endodinium simplex gave a slightly different order: palmitate, oleate, stea­ rate, linoleate. Oleate was then hydrogenated to stearate by I. prostoma. No volatile fatty acids were produced from oleate, although various fatty acids and tributyrin stimulated gas production by I. prostoma (Gutierrez et al., 1962). Williams et al. (1963) reported similar studies on I. intestinalis. In this ciliate, uptake of fatty acids was in the order: palmitate, oleate, linoleate, stearate. Other results were similar to those with I. prostoma. By such mechanisms fatty acids from the medium can be used by bacteria and protozoa to build up lipid molecules, together with fatty acids and other structures of biosynthetic origin. B.

BIOSYNTHESIS

1. General Properties of Rumen

Microbiota

A higher percentage of fats in rumen content than in the hay fed to cattle was observed by Kraus (1927), who concluded that there was net synthesis of fat in the rumen. Quitteck (1936) found essentially similar results, but concluded that most of the percentage increase in fats was due to the degradation of other components of the feed. A first indication of the general meaning of fatty acid biosynthesis in rumen has been obtained from ovines fed, for 10 days, a synthetic lipid-free, protein-free diet. In this investigation Viviani and Lenaz (1963) (Table XI) revealed the existence of very efficient biosynthesis

Table XI

Fatty acid 90 15 52 73 1045 649 1643 Traces 1727 784 Traces 143 496 458

1.17* 0.20 0.68 0.95 18.25 8.43 21.34 Traces 22.43 10.19

Traces

1.87 6.45 5.96 2.02

Milligrams of fatty acids in rumen liquor in 24 hours 6

0.69 9.06 1.65 1.77 2.20 17.20 9.34 2.48 0.64 1.01 7.98 27.16 4.36 13.93

0.55

Percent of total fatty acids in saliva D

5 72 13 14 17 138 75 20 5 8 64 217 35

Milligrams of fatty acids from saliva in rumen in 24 hours c

69 279 423

1589 709

86 15 52 68 1333 649 1629

Milligrams of fatty acids synthesized in rumen in 24 hours'*

"Viviani and Lenaz (1963). "Values are calculated by the formula: (A-B)/100, where A = total fatty acid content of rumen liquor in 24 hours (7700 mg); B = value of each individual fatty acid as percent of total fatty acids of rumen liquor. c Values are calculated by the formula (C-D)/100, where C = total content of fatty acids from saliva present in rumen in 24 hours (800 mg); D = value of each individual fatty acid as percent of total fatty acids of saliva. "Values are calculated by the formula A-B/100 - C D / 1 0 0 . ^Percentage of the total area of the gas-chromatographic elution diagram (averages of three determinations).

C 12:0 C 12:1 C 13:0 C 14:0 br C 14:0 C 15:0 br C 15:0 C 16:0 br C 16:0 C 16:1 C 17:0 br C 17:0 C 18:0 br C 18:0 C 18:1 C 18:2 Others

Percent of total fatty acids in rumen liquor B

BlOSYNTHESIZED IN RUMEN a

F A T T Y A C I D S O F R U M E N L I Q U O R AND O F SALIVA O F A S H E E P ( O V I N E ) F E D A L I P I D - F R E E D I E T , AND F A T T Y A C I D S

CD OX

to

Metabolism of Long-Chain Fatty Acids in the Rumen

296

ROMANO VIVIANI

of straight- and branched-chain C 15 acids. As for odd-chain fatty acids, the most active biosynthesis appears to be present for C 15:0. The high levels of branched-chain C 15:0 suggest that in the rumen of ovines fed a lipid- and protein-free diet, the metabolic pathways have to be extremely active leading to the formation of these fatty acids, or of precursors, which can be used for the synthesis of branched-chain C 15:0 (Table XI). Finally the authors have emphasized the presence of linoleic acid in amounts far beyond the level expected from the content of saliva present in the rumen. This latter observation can explain the reason why ruminants are not subject to essential fatty acid deficiency: in fact they always have linoleic acid of biosynthetic origin available in the rumen. The quantity of lipid synthesized by the microorganisms, as calcu­ lated by Garton (1964) is about one-fourth of the total for sheep, and that calculated by Keeney et al. (1962a) is at least 140 gm/day for the cow. Keeney et al. suggested that, as the cow yielded about 500 gm/day of butterfat, the microbial lipid could contribute substantially to the butterfat. Microorganisms isolated from rumen have contributed to knowl­ edge of biosynthetic processes of fatty acids. The biosynthetic pro­ cesses of 4 - and 5-carbon fatty acids, by incubation of 14 C-labeled ace­ tate or propionate with rumen content, have b e e n studied by Gray et al. (1951). With the use of labeled acetate, radioactive carbon ap­ peared in butyric acid and valeric acid; but with 14 C-labeled propion­ ate, valeric acid, but not butyric acid, became labeled. The mechanism of this synthesis has been studied by Elsden and Lewis (1953) with a large gram-negative coccus (L.C.) which fer­ mented certain sugars and pyruvate with the production of acetic, propionic, butyric, valeric, and caproic acids. These workers showed that the addition of either acetate, propionate, or n-butyrate to pyru­ vate fermentation increased the uptake of H 2 with an increased pro­ duction of butyrate, valerate, and caproate, respectively. Using the same microorganism, Ladd (1959), in similar experimental conditions, determined the relative specific activities and distribution of lactate2-14C in volatile fatty acids and suggested that butyrate, valerate, and caproate might be formed according to the equations of Fig. 1. Such a distribution agrees with the present knowledge of the biosynthesis of longer-chain fatty acids, such as palmitate. Nevertheless, further data that added to present knowledge of fatty acid biosynthesis in the living world in general, and in microorga­ nisms in particular (Bloch, 1962), were obtained by studies on Escher-

Metabolism

C H 8 - 1 4 C H 2 — COOH

of Long-Chain

+

Fatty Acids in the Rumen

297

2 CH3— 14COOH

+

4(H)

*- C H 3 - 1 4 C H 2 — C H 2 - 1 4 C O O H

CH 3 — 14 COOH

+

4(H)

» - C H 8 - 1 4 C H 2 — CH 2 — CH 2 - 1 4 COOH

3 CH 3 —"COOH

+

8 (H)

> - CH3— 14 CH 2 — CH2—14 CH 2 — CH 2 - 1 4 COOH

FIG. 1. Synthesis of butyrate, valerate, and caproate by rumen bacteria.

ichia coli by Bloch, Vagelos, and Wakil (Lennarz et al., 1962; Majerus et al., 1965; Wakil et al., 1964). Classic recent reviews of fatty acid biosynthesis in bacteria are those by Kates (1964), Law (1967), Len­ narz (1966), and Majerus and Vagelos (1967). Since acetyl-CoA carboxylase and the multienzyme system "fatty acid synthetase" have b e e n isolated not only from bacteria, but also from yeast and vertebrate animals, we can certainly believe that such enzymes are operative also in the rumen bacteria and protozoans. Data now available on fatty acid biosynthesis by rumen microorga­ nisms besides the nutritional volatile fatty acid (VFA) requirement concern incorporation of acetate- 14 C into long-chain fatty acids of mixed bacteria and mixed protozoans, and incorporation of amino acid- 14 C or VFA into the long-chain fatty acids of specific cellulolytic bacteria. 2. Rumen

Racteria

Cell suspensions of mixed rumen bacteria, obtained from rumen fluid, were incubated with acetate- 14 C for 3.5 hours (Viviani and Borgatti, 1967b). The long-chain fatty acid methyl esters were separated into groups according to their degree of unsaturation by column chromatography on silicic acid impregnated with silver nitrate. Aliquots were analyzed or purified by GLC, and specific activity in various fractions was measured (Table XII). The results indicate that incorpo­ ration of acetate- 14 C is mainly operative in monoenoic fatty acids; the saturated fatty acids have a lower activity. T h e small amount of ac­ tivity of C 18:2 could be due either to bacteria of another order than Eubacteriales or to contamination by small protozoans. The possible existence of rumen bacteria synthesizing linoleic acid, such as Pseudomonas aeruginosa (Romero and Brenner, 1966) appears im­ probable since in several rumen bacteria grown without lipids no C 18:2 is formed (Cunningham and Loosli, 1954; Polan et al., 1964; Vi­ viani et al., 1967b) and because synthesis of C 18:2 in Pseudomonas is aerobic, in contrast with conditions found in rumen.

C D

D

C

B

A

Table XII

Chromatographic methods

2.026 0.490

0.661

2.284

7.043

8.643

Expt. 2

1537 3200

4921

1786

1062

506

Expt. 1

850 688

860

988

466

86

Expt. 2

0.352

-

-

i



2.264

3.544

17.767

Expt. 2

1.651



2.289

2.880

14.438

Expt. 1

-

18



1609

11764

966

Expt. 1

-

82



1328

2657

503

Expt. 2

Specific activity (cpm/img)

Fatty aci(i of bacteria Weight (mg)

^The fractions A, B, C, and D represent respectively: saturated, monoenoic, dienoic, and trienoic acids

0.354 0.070

0.115

0.398

1.228

1.509

Expt. 1

Weight i(mg)

Specific activity (cpm/mg)

Fatty aci
L A B E L I N G O F F A T T Y A C I D M E T H Y L E S T E R F R A C T I O N S O F R U M E N M I X E D P R O T O Z O A AND B A C T E R I A 0

AgNOs-silicic acid column chromatography Solvent system^ Hexane-benzene (9.5:0.5 v/v), 160 ml Hexane-benzene (5:5 v/v), 120 ml Hexane-benzene (2:8 v/v), 150 ml Benzene-ethyl ether (6:4 v/v), 65 ml Gas-liquid chromatography Collection of peak: 18:2 18:3

C

<2Viviani and Borgatti (1967b).

Fr actions

14

< > 2

1—1

<

> 2 0

V O

Metabolism

of Long-Chain

Fatty Acids in the Rumen

299

Linoleic acid is normally found in lipids of mixed rumen bacteria (Section III) and also in isolated strains of rumen bacteria grown in nondelipidized media; rather than of biosynthetic origin, it must be thought to be the expression of absorption or adsorption of dietary fatty acids; it is in fact widely accepted that unsaturation in bacteria does not proceed beyond monounsaturated acids (Bloch, 1964). The suggestion, therefore, by Erwin et al. (1963) that C 18:2 in rumen might be synthesized by bacteria must be considered with caution. Among fatty acids of rumen bacterial lipids, odd- and branchedchain acids are most peculiar. Nevertheless their biosynthesis de­ pends upon the availability of "primer" structures, which often cannot be built by the same bacteria. Hungate (1950, 1957) found that two important cellulolytic bacteria, Bacteroides succinogenes and Ruminococcus sp., required growth factors present in rumen fluid but not in usual cultural media for bacteria. Bryant and Doetsch (1955) found that a branched 4- or 5-carbon chain fatty acid, plus a straight-chain acid with a 5- to 8-carbon chain, were the growth factors of rumen fluid. Palmitic or stearic acid could replace the straight-chain compo­ nent. Most strains of cellulolytic cocci Ruminococcus albus and R. flavefaciens require only the branched-chain fatty acids (isobutyric, isovaleric, or 2-methylbutyric) in combination or singly, varying among strains. One strain will grow in the absence of the fatty acids if casein hydrolyzate is present in the cultural medium, and various other strains require factors present in rumen fluid but not yet fully identified (Allison, 1965). Bryant and Robinson (1962) reported that growth of several strains oiSelenomonas ruminantium required, or was stimulated by, a mixture of volatile fatty acids that contained isobutyrate, n-valerate or isovalerate in the culture medium. Hobson et al. (1963) mentioned that ace­ tate was stimulatory for growth of a strain of Selenomonas ruminantium even in presence of a casein hydrolyzate. The addition of other volatile fatty acids, including both straight- and branched-chain acids, was not stimulatory in some cases. T h e study of Kanegasaki and Takahashi (1967) clearly demon­ strated that the strain of S. ruminantium var. lactilytica required any of the C 3:0 to C 10:0 acids at certain concentrations w h e n grown in a medium containing glucose. Among them, n-valerate was most effec­ tive at the lower concentration. In S. ruminantium also the effect of cultural medium on fatty acid composition has been investigated (Viviani et al., 1967b) (Table XIII). T h e same strain maintained on a lipid-free medium undergoes a de­ crease of palmitic and stearic acids and an increase of other acids, in

300

ROMANO VIVIANI

Table XIII EFFECT OF CULTURE MEDIUM ON FATTY ACID COMPOSITION OF Selenomonas ruminantiuma Cultural medium Fatty acid

Containing lipids

C 10:0 C 11:0 C 12:0 C 12:1 C 13:0 br C 13:0 C 13:1 C 14:0 br C 14:0 C 14:1 C15:0 br C 15:0 C 15:1 C 16:0 br C 16:0 C 16:1 C 17:0 br X1 b C 17 0 C 17 1 C 18 Obr C 18 0 C 18 1 C 19 0 C 19 1 + 18:2 C20 0 Othe rs

0.08 0.28 0.68

— Traces 1.60



0.16 2.36 2.75 0.71 8.95 8.54 0.95 10.00 8.78 0.50



4.86 1.41 2.91 11.66 5.81 11.53 2.29 7.34 5.71

Lipid-free 0.06 1.66 0.53 0.13 0.12 8.71



0.61 0.90 2.01 3.22 12.70 8.73 2.21 1.00 2.14 1.55 7.31 1.12 12.78 7.84 1.04 0.77 15.01 0.46 2.57 3.95

a

Vivmni etal. (1967b). X = unknown.

b l

particular C 15 , C 17 , C 19 , and others not yet identified. This conclusion indicates that probably saturated and unsaturated even fatty acids de­ rive in S. ruminantium from the medium, whereas odd fatty acids are typically of biosynthetic origin. T h e fermentation products vary among strains, but all produce acetic and propionic acids, some carbon dioxide, but no hydrogen. S. ruminantium appears to dispose directly of propionate from fermentation of starch as primer for odd fatty acids. T h e isotope studies by Kanegasaki and Takahashi (1967) demon­ strated that n-valerate was incorporated exclusively into lipid mate­ rials of S. ruminantium var. lactilytica.

Metabolism

of Long-Chain

Fatty Acids in the Rumen

301

In addition, in R. flavefaciens and R. albus (Allison et al., 1962), and B. succinogenes (Wegner and Foster, 1963), the intact carbon skele­ tons of the volatile branched-chain fatty acids are incorporated into higher fatty acids and in some instances into plasmalogen aldehydes. The synthesis of longer fatty acids was supposed to take place with precursors as starting blocks and subsequent addition of two carbon units to the carboxyl end of the molecules, as was described by H o r n i n g ^ a l (1960). The branched-chain fatty acids and n-valerate are produced in the rumen particularly from amino acid catabolic processes. Acetic acid, too, stimulates growth of certain typical rumen bacteria. Some rumen bacteria such as Ruminococcus flavefaciens, R. albus, Butyrivibrio fibrisolvens, and S. bovis, require biotin (Hungate, 1966, p. 81). For Bacteroides succinogenes, R. flavefaciens, R. albus, and Methanobacterium C 0 2 is indispensable, in others, such as Succinivibrio and Streptococcus bovis, it stimulates growth. The above data indicate that fatty acid biosynthesis is operative in whole rumen, but nonethe­ less it depends on a complex ecological integration of several orga­ nisms; some have enzymes necessary for fatty acid biosynthesis, but often need "primer" structures or cofactors and C 0 2 . T h e fact that some typical bacteria need up to 10-carbon straight chains could sug­ gest that some enzymes for fatty acid biosynthesis have b e e n lost. 3. Rumen

Protozoa

Viviani and Borgatti (1967b) demonstrated that after 3.5 hours of incubation with acetate- 14 C in anaerobiosis, all the methyl ester frac­ tions of protozoans are 14C labeled. In cell suspensions of sheep rumen protozoans, the fatty acids having the highest specific activities were in the order C 18:3, C 18:2, and then C 18:1. T h e saturated fatty acids had less activity. Rumen holotrich protozoans can incorporate acetate-l- 1 4 C into C 18:2 and into C 18:3 in anaerobic conditions (Table XII). The study of the position of the double bonds in unsaturated fatty acids is necessary to indicate the metabolic pathways of polyunsaturated fatty acids biosynthesis in the rumen. This problem is compli­ cated by the positional isomers derived from the biohydrogenation processes. A preliminary study on the position of the double bond of C 18:3 obtained from rumen holotrich protozoans indicates that the end product of unsaturated fatty acid biosynthesis is a-linoleic acid (Vi­ viani and Borgatti, 1968a). This observation suggests in rumen ciliates a new route for a-linoleic acid biosynthesis. In fact, according to the present opinion, the polyunsaturated fatty acid biosynthesis in higher protista and in ciliate protozoans appears to occur by an aerobic meta-

302

ROMANO VIVIANI

bolic pathway similar to that of higher animals (Hulanicka et al., 1964; Lees and Korn, 1966). The data reported above indicate that rumen protozoans may be useful in extending knowledge of the evolutionary pathways in polyunsaturated fatty acid biosynthesis (Jukes, 1966; Shaw, 1966). The biochemical mechanism of unsaturated fatty acid biosynthesis in the rumen in balance with the biohydrogenation process, could sat­ isfy the polyunsaturated fatty acid need at the rumen level and in the tissues of the ruminant. It is not known whether all rumen protozoans have mechanisms for saturated, monounsaturated, or polyunsaturated fatty acid biosyn­ thesis. Quinn et al. (1962) were able to maintain mixed cultures of rumen ciliates for periods of up 168 hours in the absence of bacteria. Various fatty acids were present in all the media, but the final one con­ tained only acetate, butyrate, propionate, and valerate, while palmitate, stearate, oleate, and linoleate were omitted. With improvements in these techniques, the requirement of protozoans for fatty acids will eventually be studied. In 1953 it was first demonstrated that ciliates require a sterol (Conner et al., 1953). Research was extended to other lipid compounds; stearate and oleate were shown to be growth factors for Paramecium (Johnson and Miller, 1958), and a relationship was found between fatty acid metabolism and cholesterol, since oleate and phosphatidylethanolamine could spare the sterol requirement of Tetrahymena setifera (Erwin et al., 1965), and since sterols were shown to be needed for unsaturated acid biosynthesis. C. B I O H Y D R O G E N A T I O N O F U N S A T U R A T E D F A T T Y A C I D S

The first demonstration of biohydrogenation was given in 1951, as already cited, by Reiser, who, after incubation of linseed oil with ruminal content, observed the transformation of linolenic to linoleic acid. In 1931 Banks and Hilditch proposed that biohydrogenation occurs in ruminant tissues, but subsequent investigations with labeled com­ pounds demonstrated that there is no biohydrogenation in animal tis­ sues. The existence of biohydrogenation processes in rumen microor­ ganisms was then the first plausible explanation of the high levels of stearic acid in the ruminant. Further studies of Reiser and Reddy (1956) showed a higher iodine number in dietary lipids than in the same lipids after they had been in the rumen for 6 hours. Shorland et al. (1955) and Hoflund et al. (1956), in studies of ruminal content, indicated an easier hydrogenation of linolenic than of linoleic acid. The problem has been studied in vivo also by means of labeled

Metabolism

of Long-Chain

Fatty Acids in the Rumen

303

compounds. After injection of linolenic acid- 14 C, w h e n the reticulum-omasal orifice was closed, about 4 5 % was completely satu­ rated. Unsaturated fatty acids were separated by paper chromatography, and the following acids were found: methyl oleate, elaidinate, linoleate, and linolenate. Only 3 - 6 % of the original labeled linolenate was still present, but its transformation into oleic and elaidic acids was 33.50% (Wood et al, 1963). 1. Studies on the Biohydrogenation

Activity

in Vitro

Whole rumen content, ruminal liquor, and rumen microorganisms have b e e n studied in in vitro experiments by several authors. Shorland et al. (1957) studied the effect of incubating oleic, linoleic, and linolenic acids singly for 48 hours with unstrained rumen content of sheep. In the presence of plant materials, about 2 0 % of each unsatu­ rated acid was also converted to stearic acid. The trans acids were formed to the extent of 17, 48, and 6 7 % from oleic, linoleic, and lino­ lenic acids, respectively. Positional isomers were produced, particu­ larly from linoleic acid, which gave rise to a conjugated form. Hydrogenation of linolenic acid does not appear to pass through cis,cis-A9'12octadecadienoic acid (linoleic acid). Under conditions similar to those of Shorland et al. (1957), the incu­ bation of ruminal liquor with linseed oil or peanut oil, whose compo­ sition in unsaturated acids simulate that of fatty acids in hay and con­ centrates, respectively, has shown formation of identical quantities of C 18:1; more 18:0 was formed from peanut oil; 18:3 isomers were formed in higher amounts after incubation with linseed oil, and 18:2 isomers predominated when peanut oil was incubated (Viviani and Borgatti, 1968b) (Table XIV). This observation suggests that the inter­ mediates of the initial steps of biohydrogenation are accumulated only for the unsaturated acid which is represented in the highest quantity in the incubation mixture. .An important consideration is that certain unsaturated C 18 acids found in ruminant fats (18:1A16trans; 18:3 cis,trans,cis; 18:3 triconjugated; all of which derive from linolenic acid) may represent specific indicators of the type of ali­ mentary fat. Studies of hydrogenating activity by various fractions from rumen began w h e n Wright (1959,1960) demonstrated that protozoans carried out complete hydrogenation to stearic acid, but that bacteria too were active. In such experiments a marked difference was observed when bacteria were suspended in phosphate buffers and w h e n they were suspended in ruminal liquor. In the latter instance, hydrogenation was much higher. To learn more about biohydrogenation by rumen

Table XIV



— 16.80* 3.29



9.09 8.89 46.76 14.27

48 Hours' incubation

0.45



6.91 2.53 28.28 58.98 2.85

Peanut oil

2.91



8.26 5.63 23.90 57.01 2.72

Zero time



19.86 c 3.93

8.33 16.02 48.43 5.74

48 Hours' incubation

Peanut oil plus rumen liquor

"Viviani and Borgatti (19686). ^Components present in higher amounts are the following: k16-trans 18:1 (tentative identification) (2.85%); trans-trans conjugated 18:2 dienes (3.94%): cis-trans and cis-cis conjugated 18:2 dienes (4.68%); cis-trans conjugated, cis isolated 18:3 (4.89%). C o m p o n e n t s present in higher amounts: trans-trans conjugated 18:2 dienes (8.45%): cis-trans and cis-cis conjugated 18:2 dienes (9.49%); cis-trans conjugated, cis isolated 18:3 (1.92%).

3.15

8.74 6.07 24.04 21.52 35.02

7.03 2.54 28.41 25.33 36.48

16:0 18:0 18:1 cis (9) + 18:1 trans 18:2 cis,cis (9,12) 18:3 cis,cis,cis (9,12,15) Total isomers of 18:1, 18:2, and 18:3 Others 0.31

Zero time

Linseed oil

Fatty acid

Linseed oil plus rumen liquor

H Y D R O G E N A T I N G A C T I V I T Y O F R U M E N L I Q U O R O N U N S A T U R A T E D F A T T Y A C I D S O F L I N S E E D O I L AND P E A N U T O I L "

2

> o < < >

2 O

Metabolism

of Long-Chain

Fatty Acids in the Rumen

305

bacteria, Polan et al. (1964) did studies using labeled linoleate in at­ mosphere of different gases. Several experiments showed that com­ plete anaerobiosis was required; hydrogenation was higher in hy­ drogen than in C 0 2 atmosphere. The authors thought this was an effect of molecular hydrogen, probably on the cell preparations, rather than its direct participation to the biohydrogenation reaction. The process was also activated by boiled ruminal liquor. Our own studies made further contributions to the localization of the hydrogenating enzymes. Fractions separated from rumen liquor by differential centrifugation show that the enzymes are contained in the microbial cells; the cell-free supernatant is not able to hydrogenate, while in all other fractions hydrogenation is effective. Complete biohydrogenation is operative only with whole liquor or with 800 g sediment, but is not carried out by washed bacteria (Viviani and Borgatti, 1967a) (Table XV). Wilde and Dawson (1966) investigated in detail the biohydrogenation process, using an artificial rumen with incubation of washed microorganisms (bacteria and protozoans) with 14 C-labeled linolenic acid. Metabolic products of C 18:3 have b e e n identified by means of thinlayer chromatography, gas chromatography, and ultraviolet and in­ frared spectrophotometry. The main pathways would be operative through a cis,cis,cis-A 9jll(13U5 -octadecatrienoic acid having two con­ jugated double bonds. At the conjugated double bonds hydrogenation occurs associated with a cis-trans isomerization of the remaining double bond. Hydrogenation of linolenic acid would not bring about formation of linoleic acid, but of cis-trans octadecadienoic compounds having two nonconjugated double bonds in the C - l l or C-12 and C-15 or C-16 positions. The following hydrogenation would produce mostly frans-monoenoic acids with double bonds mostly in C-13 or C14. Wilde and Dawson (1966), during brief incubation periods with ruminal content did not find conjugated acids. Nevertheless, after incubation of washed cells with linolenic acid, they also observed formation of conjugated acids. The presence of conjugated acids in milk (Sections VI and VII) indicates their actual formation in rumen. T h e ability of pure cultures to carry out biohydrogenation has also b e e n investigated. Polan et al. (1964) found that among 20 pure cul­ tures of rumen bacteria, only certain strains of Butyrivibrio fibrisolvens could hydrogenate linoleic acid and then only to an octadecenoic acid, but not to stearic acid. Presumably other species must exist in the rumen that are more efficient for hydrogenation and can convert the octadecenoic into stearic acid. Recent investigations on Clostridium lochheadii and

306

ROMANO VIVIANI

Table XV H Y D R O G E N A T I N G A C T I V I T Y O F W H O L E R U M E N L I Q U O R AND F R A C T I O N S

Rumen liquor plus linseed oil Incubated 24 hours

Fraction sedimented at 800 g p l u s linseed oil

Fatty acid

Linseed oil

Zero time

16:0 18:0 18:1 18:2 18:3 Total isomers of 18:2 and 18:3 Others

7.03 2.54 28.41 25.33 35.48

8^24 5.87 23.04 20.52 33.22

10.67 11.71 49.53 10.86



063 5.95 28.05 20.08 33.23



10.69 6.44

3.03



0.31

3.15

Zero time



Incubated 24 hours 9.39 8.34 48.15 16.12

— 12.83 5.16

"Viviani and Borgatti (1967a). ^Values are expressed as weight percent calculated from peak areas.

Clostridium cellobioparum (Viviani et al., 1968c) showed that cellulolytic sporogenic strains are also able to hydrogenate fatty acids (Table XVI); these strains produce H 2 , in addition to C 0 2 , acetate, butyrate, and alcohols (Matteuzzi and Crociani, 1967). Reduction of linoleic acid by a pure culture of Butyrivibrio fibrisolvens was found to produce a dienoic acid as well as a mixture of trans9,10- or 11,12-octadecamonoenoic acids. The intermediate dienoic acid leading to both monoenes is the cis,trans- or the trans,cis-9(10), ll(12)-octadecadienoic acid (Kepler et al., 1966). B. fibrisolvens was also studied by Wilde and Dawson (1966) for linoleate hydrogenation. B. fibrisolvens grown in the presence of lino­ leic acid for 21 hours partially converted it into octadecenoic acid. However, when organisms in the exponential phase of growth were incubated for 2 hours with linoleic acid under hydrogen, little hydro­ genation of the fatty acid occurred. It has been possible (Kepler et al., 1966) to separate the isomerization and the reduction steps in cell-free extracts oiB. fibrisolvens sup­ plemented with boiled rumen fluid and reduced methylviologen. T h e enzyme involved in the isomerization appears to be localized in the cytoplasm while the reductase is particulate. The isomerization pro­ ceeds in the presence of oxygen, whereas the reduction requires strict anaerobiosis. That the system is particulate is shown also by the ob­ servation that sonication destroys the hydrogenating capacity not only

Metabolism

of Long-Chain

Fatty Acids in the Rumen

307

T H E R E O F O N U N S A T U R A T E D A C I D S O F L I N S E E D OiL a f t

Fraction sedimented at 14,000 g unwashed plus linseed oil

Fraction sedimented at 14,000 g, washed, plus linseed oil

Supernatant 14,000 g plus linseed oil

Zero time

Incubated 24 hours

Zero time

Incubated 24 hours

Zero time

Incubated 24 hours

8.15 4.42 25.10 23.15 37.95

7.64 4.69 37.03 19.91 8.07

7.50 4.37 27.19 23.92 34.12

8.73 4.47 27.18 23.48 30.00

7.65 4.30 26.66 22.59 37.53

7.77 4.39 27.14 22.90 35.39

1.23

21.33 1.38

1.52

4.32 1.79

0.78

1.33 1.10

of individual bacteria, but also of rumen microorganisms (Wilde and Dawson, 1966) and of rumen bacteria (Borgatti et al., 1968). In subsequent experiments by (Kepler and Tove, 1967) incubation of B. fibrisolvens with linoleic acid produced a conjugated dienoic acid which was identified as cis-9,£rans-ll-octadecadienoicacid. When linolenic acid (cis-9,cis-12,cis-15) was used as substrate, it also was isomerized to a conjugated acid, which was tentatively identified as cis-9,£rans-ll,cis-15-octadecatrienoic acid. Subsequent hydrogenation of this conjugated trienoic acid produced a nonconjugated cistrans dienoic acid but not monoenoic acid. Incubation of whole bac­ teria with linoleic acid in D 2 0 produced cis-9,£rans-ll-octadecadienoic acid, which contained a single deuterium atom at C-13. Identification of the cis-9,trans-ll,cis-15 compound was achieved by the combination of gas-liquid chromatography and UV and IR spectroscopy. A recent experiment (Strocchi et al., 1968) (Fig. 2), employing com­ bined thin-layer chromatography (TLC) on AgN0 3 -impregnated si­ licic acid, gas-chromatography, ultraviolet and infrared spectroscopy analyses, has allowed to demonstrate that biohydrogenation of linseed oil fatty acids by rumen mixed bacteria, in presence of pyruvate as hydrogen donor yields mainly octadecatrienoic acids with two conju­ gated double bonds and small amounts of octadecatrienoic acids with three conjugated double bonds and conjugated diunsaturated acids. Conjugated dienes are mainly represented by octadecatrienoic acids having two cis-trans conjugated double bonds and one nonconjugated

ft

Vivmnietal (1968c). Values are expressed as weight percent calculated from peak areas.

0.75 10.22 0.51 5.77 24.48 22.43 33.94 1.84

14:0 16:0 16:1 18:0 18:1 18:2 18:3 Total isomers of 18:2 and 18:3

a

Zero time 1.70 11.02 0.73 7.09 25.74 27.53 16.05 9.73

Incubated 40 hours

Clostridium cellobioparum plus linseed oil

LINSEED OIL"-6

AND Clostridium

Table XVI cellobioparum

Fatty acid

BIOHYDROGENATING ACTIVITY OF Clostridium

lochheadii

Incubated 40 hours 0.60 11.67 0.65 7.85 19.82 21.52 29.54 6.82

Zero time 0.52 10.10 0.54 5.30 24.92 21.23 36.40 2.07

Clostridium lochheadii plus linseed oil

O N U N S A T U R A T E D F A T T Y ACIDS O F

2

>

> o <

O

F I G . 2. Thin-layer chromatogram of the fatty acids produced on incubation of lin­ seed oil with washed rumen bacteria in the presence of pyruvic acid. Chromatographic plate (20 X 20 cm) was covered with a 0.3-mm layer of silica gel-AgNO :5 (5:1 by weight); development of the plate: mobile phase is benzene-petroleum ether (40-70°)-ethyl ether, 70:20:10 (v/v). Fatty acid components of the individual T L C bands were identi­ fied by GLC, UV, and IR spectroscopy. A —Saturated: branched, 16:0, 17:0, 18:0; unsaturated 18:1 {trans) as impurity of the second fraction. B —Saturated: branched, 16:0, 18:0; monounsaturated: 18:1 (trans) + 18:1 (cis) as impur­ ities of fraction C; diunsaturated: 18:2 trans-trans conjugated + 18:2 cis-cis conju­ gated; triunsaturated: 18:3 di-cis-mono-trans conjugated + di-trans-mono-cis con­ jugated + tri-trans conjugated. C —Monounsaturated: 18:1 cis+ 18:2 cis-trans, and cis-cis conjugated, present in traces as impurities from the second fraction. D —Monounsaturated: 16:1 cis, 18:1 cis (from the previous fraction). Diunsaturated: geometric isomers of linoleic acid with isolated cis-trans and trans-cis double bonds. Triunsaturated: conjugated tri-cis trienes. E —Diunsaturated: 18:2 cis-cis as impurity from the sixth fraction. Triunsaturated: diconjugated trans-trans with one isolated trans double bond + diconjugated cistrans with one isolated trans double bond. F - D i u n s a t u r a t e d : 18:2 (9-cis,l2-cis). G — Diunsaturated: 18:2 (9-cis,12-cis) as impurity of the previous fraction; triunsatur­ ated: conjugated dienes: cis-trans conjugated with one cis isolated (impurity of the third fraction) + trans-trans conjugated with one cis isolated (IR + UV). H —Triunsaturated: conjugated dienes: cis-trans conjugated with one cis isolated (IR + UV). I —Triunsaturated: linolenic acid (9-cisy12-cis,15-cis).

310

R O M A N O VIVIANI

cis double bond in the band T L C H, and by octadecatrienoic acids having two conjugated trans double bonds and one isolated cis double bond present in the band TLC C Smaller amounts are present of octadecadienoic acid with a trans-trans conjugated configuration (TLC band B). Conjugated trienes also are present in the second TLC band, and their configurations are mainly di-trans, mono-cis and to a lesser extent di-cis, mono-trans; and tri-trans. It is likely that also a conju­ gated triene having three cis double bonds is present (band T L C D). Geometric isomers of linoleic acid with cis-trans or trans-cis double bonds are also present in the T L C band D in traces. Traces of monounsaturated trans acids are present in the second TLC band, and also as impurity in the first TLC band (Fig. 2). From the data reported above w e can tentatively draw an overall scheme of the biohydrogenation process (Fig. 3). The results of Kepler and Tove (1967) and of Strocchi et al. (1968) on formation of 18:3 cis-trans conjugated, cis isolated by B. fibrisolvens and by rumen bacteria, agree with that observed in the first phase of the alkali isomerization process of linolenic acid, which is accomLINOLENIC ACID (cis-cis -cis - Nonconj ugated octadecatrienoic acid)

Triconj ugated octadecatrienoic acids

trans - trans - Conj ugated cis - nonconj ugated octadecatrienoic acid

cis - trans - Conj ugated, cis- nonconj ugated octadecatrienoic acid

cis-cis - Conj ugated, cis -nonconjugated octadecatrienoic acid + 2H

\+2H

\

cis -cis- Nonconj ugated octadecadienoic acids

+ 2H cis- trans - Nonconj ugated octadecadienoic acid

LINOLEIC ACID ■ (cis-cis- Nonconj ugated octadecadienoic acid)

trans - trans (or cis-trans \ or cis-cis) + 2H Conjugated > trans- Octadecenoic acid octadeca­ dienoic acid + 2H

OLEIC ACID +2H - - (czs-Octa- -« 1 decenoic acid)

STEARIC ACED F I G . 3. ganisms. (1966).

Pathways of biohydrogenation of unsaturated fatty acids by r u m e n microor, Kepler and Tove (1967), Strocchi et al (1968); , Wilde and Dawson

Metabolism

of Long-Chain

Fatty Acids in the Rumen

311

plished heterolytically (Nichols et al., 1951; Strocchi, 1968) and also with the autoxidation process of the same acid, which occurs with the homolytic mechanism (Holman, 1954). Shift of a double bond toward the conjugated positions occurs, with formation of octadecatrienoic acids with two conjugated cis-trans double bonds and one isolated cis double bond. The double bond which shifts, passes from the cis configuration, almost completely to the trans configuration, according to the prototropic mechanism of Nichols et al. (1951). On the other hand, the formation of cis-cis conju­ gated, cis isolated octadecatrienoic acid (Wilde and Dawson, 1966) cannot be explained according to these chemical principles. T h e small quantity of triconjugated octadecatrienoic acids in the rumen (Strocchi et al., 1968) is in accordance with the second phase of the alkali isomerization process. Fatty acids with a cis isolated double bond, separated from the diene conjugated system by only one methylenic group, give rise to octadecatrienoic acids with three conju­ gated double bonds mostly in the di-trans-mono-cis configuration. T h e results of Kepler and Tove (1967) on linoleic acid agree with Nichols' principle (see Nichols et al., 1951), for formation of conju­ gated cis-trans diene. The significant amount of cis-trans nonconjugated octadecadienoic acids observed by Wilde and Dawson (1966), in rumen hydrogenation could be due to hydrogenation of one of the two conjugated double bonds of cis-trans conjugated, cis isolated octa­ decatrienoic acid. Furthermore, conjugated cis-trans octadecadienoic acid may follow the pathways already shown for linoleic acid, i.e., hydrogenation with formation of trans C 18:1. From the cis-trans nonconjugated octadecadienoic acid, according to the chemical principles previously considered, trans-trans conju­ gated octadecadienoic acids may be formed and to a lesser extent cistrans conjugated and traces of cis-cis octadecadienoic conjugated acids. Such octadecadienoic conjugated acids may then be hydrogenated. The fact that in experiments with linseed oil incubated with rumen bacteria (Strocchi et al., 1968) mostly trans-trans conjugated acid is formed, allows us to consider a metabolic pathway passing through trans-trans conjugated compounds and which then by hydrogenation brings about trans 18:1 formation. 2. Hydrogen-Donating

Metabolites

If much is yet to be understood of the metabolic pathways, there is

312

ROMANO VIVIANI

almost no knowledge of the hydrogen sources that are necessary for the biohydrogenation process. It is well known that rumen is an active reducing biological envi­ ronment. The contents of the rumen are very highly reductive. The Eh measured between electrodes of platinum and calomel usually lies between—360 and—420 mV (Howard, 1967). Besides reductive deamination of amino acids, methane formation from C 0 2 , formation of propionic and lactic acids, there is in rumen an active hydrogenation of unsaturated fatty acids. The problem of hydrogen donors may be studied on the basis of present knowledge of metabolic processes in rumen that can produce molecular hydrogen. In the complex rumen ecology, sometimes a symbiosis of different microorganisms brings about a breaking down of pentoses and hexoses to three-carbon com­ pounds, and then to terminal products; at other times, the whole pro­ cess is the work of one microorganism. In fact, there are microorga­ nisms that transform pentoses and hexoses into acetate and formate, and other organisms that transform them into acetate, C 0 2 , and a re­ duced compound, such as H 2 , succinate, propionate, or butyrate. There are also microorganisms capable of producing more or less re­ duced terminal compounds. Metabolic pathways for reductive processes and H 2 production appear to be associated with the mechanisms of pyruvate breakdown. Two of these processes appear to be operative in rumen: (1) the clostridium phosphoroclastic (thioclastic) type; (2) the coli-aerogenesphosphoroclastic (formate phosphoroclastic) type (Baldwin, 1965). The first system requires CoA, Fe 2 + , and thiamine pyrophosphate as cofactors; it catalyzes oxidative decarboxylation of pyruvate to form acetylphosphate, C 0 2 , and H 2 (Fig. 4). This clostridium-phosphoroclastic system has been extensively studied and characterized in Clostridium butyricum (Mortlock et al., 1959) and in Peptostreptococcus elsdenii (Peel, 1960). The first elec­ tron acceptor of the thioclastic system is a low molecular weight pro­ tein containing iron and denominated ferredoxin (FD). Ferredoxin is the electron transfer protein having the lowest known redox potential (Mortenson et al., 1963). The second type of a phosphoroclastic system, also known as the formate phosphoroclastic system, requires CoA, Fe 2 + , thiamine pyro­ phosphate, and probably Mn 2 + or Mg 2+ . The products of pyruvate deg­ radation are acetylphosphate and formate (Fig. 4). The formate phos­ phoroclastic reaction has b e e n characterized in Veillonella gazogenes, in alkaline conditions; it seems to be the characteristic type of fermen­ tation of some cellulolytic microorganisms since their final products

Metabolism

of Long-Chain

Overall reaction: C H 3— C O - C O O H

+ H 20 =-»-C02

+

H2

Fatty Acids in the Rumen +

313

CH 3—COOH

C l o s t r i d i a l p h o s p h o r o c l a s t i c (thioclastic) s y s t e m H J P 0 4, D P T | 1 C H 3 — C O - C O O H + CoASH + F D — ^ - CO a + C H 3 — C O ^ S C o A -+ FDH 2 2+ Fe ' ' _ . ., Ferre, , ~ . Reduced A Acetyl-CoA Pyruvicacid nd o x i £ e r r e d on x i (a)

ff C H 3— C O ^ S C o A + H 3P 0 4

FDH 2

" \ + NAD Reduced ^ - ^ ^ ferredoxin ^ NADH + H + + F D (b) O OH C H 3— C O ^ P = 0 3 \^

/

+

/ 0H

»~ C H 3 — C O ^ P = 0 + CoASH X OH

Acetyl-CoA

Acetyl-P (c)

ADP

*-

I

CH 3—COOH + ATP 3 1

OH Acetyl-P

Acetic acid (d)

Overall reaction: CH3— CO-COOH

+ H20 — » - HCOOH

+

CHg—COOH

Formate phosphoroclastic system

CH3—CO—COOH + H 3 P0 4

CoASH DPT * " »- 1 HCOOH 1 + C H 3 — C O ~ P = 0 X Mn2+, Fe 2 + OH

Pyruvic acid

Formic acid

Acetyl-P

(a) 9

.OH 1 +

ATP

OH Acetyl-P

Acetic acid (b)

F I G . 4.

Possible mechanisms for the decarboxylation of pyruvate in the rumen.

include formate but not hydrogen (McCormick et al., 1962). Formate deriving from phosphoroclastic breakdown of pyruvate is rapidly transformed in rumen into C 0 2 and CH 4 , probably through the pro­ cesses (Peck et al., 1957; Brill et al., 1964) reported in Fig. 5. A micro­ organism utilizing formate or H 2 in rumen is Vibrio succinogenes (Wolinetal.,1961). Our researches (Viviani et al., 1967c) have contributed to solving

314

ROMANO VIVIANI

H-COOH

+

Formic acid

formic

FD

^- co2

dehydrogenase

Ferredoxin

+

FDH2 Reduced ferredoxin

(a)

+ NAD

-*- FD + NADH

+ H

FDH2 -*- FD + H2

? Hydrogenase

(b)

F I G . 5.

Possible mechanisms for formate degradation in the rumen.

the problem of hydrogen donors and of their role in biohydrogenation. The phenomenon has b e e n studied in vitro in an artificial rumen; w e have used gas-chromatographic analysis of fatty acid methyl esters before and after incubation for 48 hours in a C 0 2 environment at a

Table XVII E F F E C T O F VARIOUS M E T A B O L I T E S AS H Y D R O G E N DONORS F O R BIOHYDROGENATION O F UNSATURATED FATTY ACIDS

Fatty acid composition of incu nation system Zero time

Fatty acids C 16:0 C 18:0 C 18:1 C 18:2 C 18:3 Dienoic isomers Trienoic isomers Total isomers Other

8.44 5.13 26.48 23.55 34.99

— — —

1.79

After 48 hours of incubation

No addition

Glucose 55.5 mM

Pyruvic acid lllmM

Pyruvic acid 55.5 m M + glucose 27.8 mM

8.73 4.47 27.18 23.48 30.00

7.96 4.93 27.88 24.04 24.04 1.98 6.93 8.91 1.72

7.64 5.87 29.90 22.63 17.52 5.79 8.77 14.56 1.87

8.23 5.46 27.03 24.07 20.56 2.13 10.04 12.17 2.52



4.32 4.32 1.79

"Viviani etal. (1967c). ^Values are expressed as weight percent calculated from peak areas.

Formic acid 333 mM 7.83 5.37 28.60 24.38 28.76 1.45 1.26 2.71 2.33

Metabolism

of Long-Chain

Fatty Acids in the Rumen

315

constant p H of 6.6: the disappearance of polyunsaturated acids, and the appearance of positional and geometric isomers of 18:2 and 18:3, were calculated. T h e process was then investigated in washed cells of mixed ruminal bacteria, suspended in "artificial saliva." In such conditions several substrates were tested as hydrogen donors for the biohydrogenation of dietary linoleic and linolenic acids. Among the various metabolites tested (glucose, pyruvate, formate, succinate, a-ketoglutarate, acetate, propionate, and mixtures thereof), pyruvate or formate in association with glucose have notably increased the hydrogenating activity: suc­ cinate and a-ketoglutarate exerted a partial increase; acetate and pro­ pionate had little effect (Viviani et al, 1967c) (Table XVII). The action of pyruvate and formate agrees with the idea that they represent the most direct hydrogen precursors. The partial action of succinate and a-ketoglutarate may be explained by their transforma­ tion into pyruvate. The highest activity of pyruvate on the washed bacteria of the 14,000 g fraction might indicate a more active thioclastic process in association with the first biohydrogenation steps. In any case, those observations suggest that detailed studies are n e e d e d of the processes of phosphoroclastic system of pyruvate and

O F L I N S E E D O I L BY W A S H E D M I X E D R U M E N BACTERIA"'"

Fatty acid composition of incubation system After 48 hours of incubation Formic acid 156 mM+ glucose 27.8 mM

Succinic acid 37 mM

7.15 4.21 27.71 25.25 24.91 2.63 4.07 6.70 2.78

8.17 5.05 23.26 23.39 27.66 2.04 7.54 9.58 2.89

glucose 27.8 mM

a-Ketoglutaric acid 64.7 mM

a-Ketoglutaric acid 32.4 m M + glucose 27.8 mM

Acetic acid 166.5 mM

Propionic acid 111 mM

7.45 4.71 27.34 24.53 24.33 2.24 7.51 9.75 1.86

8.48 4.99 29.58 22.83 22.20 1.73 8.21 9.94 2.01

8.92 5.37 29.74 27.06 23.63 1.07 1.13 2.20 3.09

8.95 4.61 25.05 22.48 29.08 2.95 5.52 8.47 2.35

8.31 4.54 26.85 23.87 28.84 2.08 3.28 5.36 2.25

Succinic acid

37mM+

316

ROMANO VIVIANI

breakdown of formate in bacteria to produce the hydrogen necessary for biohydrogenation. In addition, the observation that the ruminal protozoans Isotricha intestinalis and I. prostoma hydrogenate oleic to stearic acid (Wil­ liams et al., 1963) might also indicate participation of protozoans in the complete hydrogenation. On the other hand, the most recent ob­ servations by Katz and Keeney (1967) and Viviani et al. (1968a) on the low content of stearic acid in neutral fat and nonesterified fatty acids of protozoans appear to indicate a low hydrogenating activity of these organisms. 3. Unknown

Factors Required for Complete

Hydrogenation

Polan et al. (1964) reported that boiled rumen fluid stimulated the hydrogenation of linoleic and oleic acids from rumen microorganisms of a steer, but attempts to substitute for ruminal liquor with compo­ nents such as glucose, formate, or amino acids were not successful. The full hydrogenation capacity of rumen microorganisms toward alinolenic and oleic acid requires, according to Wilde and Dawson (1966), an essential cofactor that is present in the rumen liquor super­ natant and cannot be substituted for by NADH, succinate, lactate, or glucose. Such experiments on a single fatty acid indicated that washed cells of microorganisms could not operate the entire hydrogenation pro­ cess. Viviani and Borgatti (1967a) showed that washed bacterial cells hydrogenate only with rumen liquor added, and they indicated (Vi­ viani et al., 1967c) the activity of metabolites, such as glucose, pyruvate, formate, on the biohydrogenating process of washed bacterial cells. Several explanations may be given as to the apparent discrepancy between our findings and those of others. In the first place, Wilde and Dawson (1966) did not use bacteria, but whole rumen microorga­ nisms, and both time and hydrogen donor concentration were insuffi­ cient; on the other hand, Polan et al. (1964) used hydrogen, which could substitute for hydrogen-donating substrate. This means that investigations of unknown factors required for hydrogenation, present in ruminal liquor, first need consideration if they are hydrogen donors or cofactors for the system. To exclude the influence of hydrogen do­ nors, it is first of all necessary to saturate the system with hydrogen donors (Viviani et al., 1967c) so that they can meet all reduction re­ quirements: methane formation from C 0 2 , and other processes. Only

Metabolism

of Long-Chain

Fatty Acids in the Rumen

317

in such a way can the existence of other factors be investigated: they could be coenzymatic in nature, involved in various metabolic steps. We cannot forget that the microbial material represents a particular ecology; only the symbiosis of different microorganisms appears to render the whole hydrogenation process operative. 4. Biochemical

Significance

of

Biohydrogenation

Because of their considerable amount, unsaturated fatty acids in rumen can represent an "electron sink" and could be an alternative pathway of other reductive processes for electrons. The heats of hydrogenation of unsaturated compounds deserve at­ tention. Heat of hydrogenation may be defined as the enthalpy change when 1 mole of one unsaturated hydrocarbon is transformed into the corresponding saturated compound (Kistiakowsky, 1936; Glasstone, 1956). For ethylene the value is AH = —32.58 kcal., and the sign is for heat liberation. In the hydrogenation processes, i.e., when unsaturated compounds become saturated, there is then energy liber­ ation. Is this energy utilized? It is possible that certain hydrogenating microorganisms utilize such energy as ATP for doing work, for syn­ thesis of new organic matter. In addition, stearate, which is a typical metabolite for ruminants, represents from the energetic point of view a better energy source, since the heats of combustion are higher for saturated than for unsaturated acids (Czerkawski et al., 1966a). D.

OTHER

PROCESSES

1. Fatty Acid

Catabolism

Bauchop and Elsden (1960) have shown a fairly precise relationship between the amount of bacterial cell material synthesized and ATP made available by the fermentation of an energy source. Although hexose and pentose represent the main energetic sources, long-chain fatty acids are also potential energy sources, which may yield high amounts of ATP through the catabolic degradation process, as in the animals and higher plants. Fatty acid degradation might have signifi­ cance, since rumen bacteria are found in a growth stationary phase (Section I). Duncombe and Glascock (1954) observed that when tritium labeled stearic acid was fed to a goat, labeled lower fatty acids were found in the milk fat, indicating catabolism not only in tissues, but also in rumen. Experiments in vitro with rumen content (Garton, 1961) and with bovine rumen bacteria (Polan et al., 1964) and in vivo, with 14 C-

318

ROMANO VIVIANI

labeled linolenic acid introduced in rumen of sheep in which the reticulum-omasal orifice was ligated (Wood et al., 1963), indicated that long-chain fatty acids were not appreciably degraded. An active oxidation process of long-chain fatty acids having impor­ tance in the complex energetic economy of ruminants does not, there­ fore, seem to be operative in the rumen. Catabolism of long-chain fatty acids then does not appear to be an important mechanism for ATP production in the rumen. Moreover, in investigations on the signifi­ cance of lipids in bacteria, Lennarz (1966) does not believe that they could be energetic sources, as for higher plants of animals, and states that energy production is not the primary function of the lipids in Eubacteria. As for protozoa, we must consider that the renewal of rumen content does not allow them a stationary growth phase; lipids therefore should be mainly used as a source of synthesis of protoplasmic material. Oxidation should not be important then, although the lipids could be, as in higher organisms, not only structural components, but also energy depots (Dewey, 1967; Shaw, 1966). 2. Ketoacid

Biosynthesis

The presence of ketostearic acids in milk fat (Keeney et al., 1962b) and in the rumen (Katz and Keeney, 1967) —where they comprise about 45 micromoles per gram of lipid and have the keto group in po­ sition 16(73%), 13 (8.8%), or 9 ( 6 . 2 % ) - h a s advanced the problem, yet to be solved, of their metabolic origin in rumen; in fact hydroxy and keto acids have never been found in the forages of those animals in whose rumen these compounds were isolated. In investigations unrelated to rumen, Wallen et al. (1962) reported the possibility of Pseudomonas producing 10-hydroxystearic acids from oleic acid in an overall yield of 14%. Preliminary experiments of Keeney and Katz indicate relationships between keto- and hydroxystearic acids in the rumen. When 12-hydroxystearic acid was incu­ bated in vitro with rumen content, two-thirds of the recovered radio­ activity was found in ketostearic acid. Although Boyd et al. (1965) reported that the incubation of linoleic acid-l- 14 C with rumen digesta did not result in the production of la­ beled ketostearic acid, and although Katz and Keeney (1967) indicated that oleic-l- 14 C and linolenic-l- 14 C acids are converted to ketostearic acid in a very limited amount (0.5% of added radioactivity), the possi­ bility remains that unsaturated fatty acids are converted to hydroxyand successively to ketostearic acid in the rumen.

Metabolism

of Long-Chain

3. Aldehyde

Fatty Acids in the Rumen

319

Formation

Considering that certain bacteria and protozoans typical of rumen contain fatty aldehydes, which are absent in vegetables, it appears that transformation of an acid to the corresponding aldehyde is opera­ tive in these microorganisms (Sections III and IV). Certainly, in a reductive environment such as the rumen, this transformation seems likely. 4. Biosynthesis of Multibranched Fatty Acids Formation of phytanic or pristanic acids may result from hydrogenation of the double bond followed by oxidation of the alcoholic group of phytol of chlorophyll according to the scheme in Fig. 6. E.

METABOLIC

CONTROL

The control of lipid synthesis in bacteria is an important mechanism about which we know very little. The same is true concerning this process in rumen microorganisms. In animals, control of biosynthesis is exerted through acetyl-CoA carboxylase (Majerus and Vagelos, 1967; Lynen, 1967); the bacterial enzymes do not seem to respond similarly. A control point should be the /3-hydroxydecanoyl-CoA dehydrase for the proportion of saturated and unsaturated fatty acids (Norris et al., 1964). This could be impor­ tant also in the economy of rumen, where unsaturated acids act as electron acceptors. Availability of amino acids seems to affect mem­ brane lipids. Deficiency of certain essential amino acids induces lipid accumulation (Shockman et al., 1963). This, too, could be a control mechanism, in fact it seems reasonable that utilization of an appro­ priate protein to make up a given lipoprotein structure, could be im­ portant in the control of fatty acid synthesis in bacteria. Phytol (3, 7,11,15-tetramethyl2-hexadecen-l-ol)

Pristanic acid (2,6,10,14-tetramethylpentadecanoic acid)



Phytanic acid (3, 7,11,15-tetramethylhexadecanoic acid)

FlG. 6. Conversion of phytol into multibranched fatty acids in the rumen (Hansen, 1966; Patton and Benson, 1966).

320

ROMANO VIVIANI

As for antibacterial agents, we have demonstrated (Viviani et al., 1967d) that the antibiotics Aureomycin and chloramphenicol, intro­ duced directly into the rumen, lower the total fatty acid content of rumen and lower also the number of bacterial cells (as shown by the lower DNA levels). Aureomycin lowers the percentage of branched and odd fatty acids. It is possible that the antibacterial action of Aureo­ mycin is effective not only on microorganisms producing volatile fatty acids, but also on the microorganisms utilizing such volatile acids as precursors of the synthesis of odd and branched long-chain fatty acids. In fact, it is known that Aureomycin in rumen, besides inhibiting the production of propionic acid (precursor of odd-chain fatty acids) (Conrad et al., 1958), induces a decrease in cellulose breakdown (Barnett and Reid, 1961, p. 94). The process of cellulose breakdown is effected by bacteria to which volatile branched fatty acids are essen­ tial growth factors (Section IV, B); odd and branched long-chain fatty acids are characteristic components of cellular lipids in such bacteria (Section III). Moreover, streptomycin does not affect the total lipid content, but modifies the stearic and oleic acid contents (Viviani et al., 1967d). Some sulfonamide compounds inhibit the biosynthesis of branched-chain fatty acids (Viviani et al., 1963b). In Protozoa, control has been studied only in investigations unre­ lated to rumen, and it was shown that inhibition of fatty acid synthesis by triparanol (which also inhibits protozoal growth) is removed by administration of unsaturated fatty acids (Dewey, 1967). Unsaturated acids also appear to counteract inhibitory action by polyene antimycotic antibiotics (Dewey, 1967). V.

Relationships between Long-Chain Fatty Acids and Some Metabolic Processes of Rumen Microorganisms

A. L O N G - C H A I N F A T T Y A C I D S AND C E L L U L O S E D I G E S T I O N

Brooks et al. (1954) noted inhibition of cellulose digestion in rumen by maize oil; the inhibitory effect of the oil was counteracted by the inclusion of alfalfa ash. A stimulation of cellulose or fiber digestibility by this material had previously been recorded. Ward et al. (1957) have shown that alfalfa ash counteracts the depressive action of corn oil on fiber digestibility when oil is less than 5 % of the total ration; a trace mineral supplement based on the composition of alfalfa ash was equally effective for cellulose digestion (Summers et al., 1957); White et al. (1958) found that supplementation with 5 % corn oil decreased cellulose digestibility progressively for 40 days, and that digestibility

Metabolism

of Long-Chain

Fatty Acids in the Rumen

321

was not restored until 17 days after removal of the oil. Addition of cal­ cium, or calcium plus phosphorus, to the fat-supplemented ration could simulate the effect of alfalfa ash. Other experiments on this sub­ ject are reported by Davison and Woods (1963) and Roberts and McKirdy (1964). Buysse (1962) showed in sheep that the depression was evident only w h e n more than 30-45 gm of fat was added to the diet. This represents about 5 % of the diet, and it is generally accepted that amounts of fat greater than about 7 % of the diet are not tolerated by ruminants. The available data do not allow us to understand whether inhibition is due to action on specific enzymes or on microorganisms. Among several inhibitors tested on the specific enzymes, lipids or fatty acids were inactive, although anions were shown to be able to inhibit; betaine, among lipid constituents, is inhibitory (Mandels and Reese, 1963). The marked depression of cellulose digestion reflects an effect of fatty acids, rather than of glycerides. Czerkawsky et al. (1966a) studied the comparative effects of linseed oil and of linseed oil fatty acids in­ corporated in the diet on the metabolism of sheep, and observed a fall in cellulose digestion. B. L O N G - C H A I N F A T T Y A C I D S AND M E T H A N E

PRODUCTION

It has b e e n estimated that the ruminant may lose 10% of its diges­ tible nutrients in the form of gas. This gas consists mainly of carbon dioxide (65%), methane (27%), and 7 % of nitrogen; traces of 0 2 (0.6%) of hydrogen (0.2%), and hydrogen sulfide (0.01%), are also present (McArthurandMiltimore, 1961). T h e composition of rumen gas has been shown to vary regularly with the feeding cycle (Kingwill et al., 1959). The ratio of carbon dioxide to methane was narrowest (1:1) after fasting and widest (3:1) within 2 hours of feeding. The ratio narrowed until a secondary wide peak appeared 4 hours after feeding, then gradually narrowed again to that of the fasting level. The nature of the ration did not appear to have a very marked effect on the changes in composition of the rumen gas. Reduction of C 0 2 provides the main source of methane in the rumen (McNeill and Jacobson, 1955), hydrogen being the limiting factor in the reaction. The results were confirmed by Smith and Hungate (1958) with methanogenic bacteria isolated from the rumen. Ac­ cording to Carrol and Hungate (1955) and Vercoe and Blaxter (1955), 1 mole of formic acid gives rise to 0.25 mole of CH 4 . Methane may be formed by reduction of C 0 2 from sources other than formate (Opper-

322

ROMANO VIVIANI

mann et al., 1957; Nelson et al., 1958), such as acetate, butyrate, or valerate. It is possible that hydrogen acceptors, other than C 0 2 , present in the rumen might compete with C 0 2 and decrease methane produc­ tion. Of the competing substances, unsaturated fatty acids have been considered and found to be effective in inhibiting methanogenesis (Czerkawski et al., 1966b). Since inhibition of methanogenesis is not accompanied by a similar serious parallel inhibition of cellulolysis, Czerkawski et al. (1966b) concluded that the inhibitory effect of unsaturated acids is not at the level of cellulolytic or amylolytic bacteria which produce the terminal metabolites utilized by methanogenic organisms, but is effected on the latter organisms only. Demeyer and Henderickx (1966) showed that washed cell suspen­ sions of sheep rumen bacteria produce C 0 2 , CH 4 , acetic, propionic, and butyric acids, from sodium pyruvate. Results obtained with carboxyl-labeled pyruvate suggest that metabolic hydrogen is formed by the reaction shown in Fig. 7. This reaction is in accordance with the pyruvic phosphoroclastic process. This hydrogen is used for produc­ tion of methane, propionic acid, and butyric acid according to the re­ actions shown in Fig. 8. It was shown that hydrogen is used predominantly in both methane and propionic acid production and to a lesser extent in butyric acid production. It can easily be imagined that the individual amounts of the end products formed are the results of a competition between dif­ ferent bacteria (or substrates) for available metabolic hydrogen. Among the various compounds, theoretically able to compete for metabolic hydrogen in order to lower methane production, higher unsaturated fatty acids were tested. The percent distribution of hy­ drogen clearly showed that unsaturated fatty acids produce a consid­ erable shift in hydrogen utilization toward propionic acid, at the ex­ pense of methane and butyric acid. An inhibition of methane production from formate was also observed in presence of unsaturated acids. A direct toxic effect upon methane bacteria is involved. Stimulation of propionic acid production could be explained as the indirect conseCHS—CO—COOH Pyruvic acid F I G . 7.

+H2

° »

CH3—COOH

+

COz

+ H2

Acetic acid

Hydrogen production from pyruvate by mixed rumen bacteria.

Metabolism

of Long-Chain

Fatty Acids in the Rumen

(CO,/ + 8 H

CH 3 —CO-COOH + 4 H

2 CH3—COOH + 4 H

»- CH4 +

323

2 H20

+- CH3—CH2—COOH

+

HzO

*- CH3—(CH2)2—COOH + 2 H 2 0

F I G . 8. Hydrogen utilization in the production of methane, propionic acid, and bu­ tyric acid by rumen bacteria (Demeyer and Henderickx, 1966). (C0 2 ) + stands for free carbon dioxide as well as for a carboxyl group or other bound form.

quence of this effect. Nevertheless a direct effect of unsaturated fatty acids upon propionic acid production cannot be excluded. The effect of unsaturated fatty acids upon in vitro methane and propionic acid production can explain the related in vivo results by Robertson and Hawke (1964). Also the effect of positional and geometrical isomers has b e e n considered. Methane production is not in itself a wasteful process; the free en­ ergy change associated with the reduction of C 0 2 is —31 kcal/mole, and thus the formation of methane permits the synthesis of microbial material which becomes available to the host. A bacterium (M. ruminantium) which obtains energy by means of the above reaction or from formic acid was isolated by Smith and Hungate (1958). T h e biohydrogenation process may have bioenergetic significance (Section IV). The problem can arise from these considerations, whether unsaturated acid hydrogenation can produce in rumen more material than does methanogenesis. In fact, in methane formation the energy of the transformation of C 0 2 to CH 4 is made available for pro­ tein synthesis, nevertheless the final product cannot be used by the animal, being eliminated in the form of gas. On the other hand, reduc­ tion of unsaturated acids not only could make energy available, but the terminal product, i.e., stearate, also is directly and better absorbed and used by the host (Section IV), and with a higher caloric gain than the homologous unsaturated fatty acids. C.

U N S A T U R A T E D F A T T Y A C I D S AND O T H E R R E D U C T I V E PROCESSES

Another reductive process in the rumen is the acrylic acid pathway. Acrylate derives from lactate and is reduced to propionate. Addition of acrylic acid was found to inhibit fatty acid hydrogenation, probably by hydrogen competition (Polan et al., 1964). Other reductive reactions

324

ROMANO VIVIANI

in rumen are transformation of nitrates to nitrites, sulfates to sulfites, riboflavin to leucoflavin (McNeill and Doetsch, 1956); the coupled deamination of two amino acids, one of which functions as hydrogen donor and the other as a hydrogen acceptor (el-Shazly, 1952). Further­ more the insecticide parathion is reduced to p-amino compounds (Cook, 1957). Until now, none of these processes has been studied in relation to unsaturated fatty acids. It is possible, however, that also in the case of these substrates there may be competition both with the hydrogenation process and with methane production. Methane pro­ duction itself could be studied by using these substrates, possibly in association with unsaturated fatty acids. Moreover, the use of methanogenesis inhibitors might be useful for a study of the various "electron sinks" in rumen, such as unsaturated fatty acids.

VI.

Fate of Rumen Long-Chain Fatty Acids

A. A B S O R P T I O N F R O M T H E D I G E S T I V E

TRACT

While rumen mucosa absorbs volatile fatty acids, long-chain fatty acids are absorbed predominantly in the small intestine. Such demon­ strations were also obtained by investigations in which linoleic acid14 C was introduced into a sheep rumen, after the reticulum-omasal orifice had been bound. In the mucosa and the muscular tissue of rumen, the activity is 0.3-0.5% of the total dose, representing the quota absorbed by the ruminal epithelium. In liver, lung, kidney, and spleen the activity ranges between 0.1 and 0.001%; it is higher in liver, however. Since some radioactivity is observed in the volatile fraction of blood, and activity is also present in the volatile fraction of rumen content, a degradation of labeled linoleic acid in the rumen has to be involved (Wood et ah, 1963). Radioactivity remaining in rumen ranges between 85 and 9 6 % of the total amount. An interesting aspect appears to be the demonstration that the rumen wall is capable of transforming hydrocarbons present in the vegetables into fatty acids by co-oxidation mechanisms (McCarthy, 1964). The main mechanism of lipid absorption is localized in the intes­ tinal tracts below the rumen. It has b e e n calculated that, on a hay diet, an ovine absorbs about 25 gm of lipids in 24 hours, 7 gm of which, ac­ cording to Viviani and Lenaz (1963), may be of biosynthetic origin. Most of them are free fatty acids deriving from ruminal lipolysis, in contrast with what happens in the monogastric animal. Nonetheless, free fatty acids are emulsified by bile salts, perhaps in association with

Metabolism

of Long-Chain

Fatty Acids in the Rumen

325

lysophosphatidylcholine of biliary origin. If a ruminant is deprived of bile and pancreas, there is no intestinal absorption of lipids (Heath and Morris, 1963). Bile lipids are rich in phospholipids, and it is be­ lieved that 10-15 gm enter the small intestine of sheep every day (Adams and Heath, 1963) and mix with those of ruminal origin. This could explain the higher levels of unsaturated acids found in the intes­ tinal content than in rumen (McCarthy, 1962a). A distinguishing fea­ ture of the ruminant is also the high amount of stearic acid absorbed (5.6-7.4 gm in sheep in 24 hours); in ruminants there is an evident fa­ cility for stearic acid absorption (Felinsky et al., 1964); whereas the monogastric animal (rat) has a digestibility coefficient for stearic acid of only 2 4 % (Carroll and Richards, 1958). According to Ward et al. (1964), trans C 18:1 acids deriving from rumen are almost quantitatively absorbed by ileum. In lipid metabo­ lism in the large intestine ruminants also differ from monogastric ani­ mals. Unsaturated acids present in ileum are hydrogenated in the cecum and the colon with formation of trans C 18:1 and its absorption. The intestinal absorption of certain particular fatty acids present in the lipids of some vegetables, such as eleastearic, ricinoleic, or erucic acids, has been investigated by direct introduction into the duodenum (Felinska, 1966). B. U T I L I Z A T I O N F O R R U M I N A N T L I P I D S

As in the monogastric animal, absorbed lipids reach blood circula­ tion through the thoracic duct, where stearic acid is the most abundant component, and reflect the metabolic conditions in rumen, rather than the composition of the intestine. In chylomicrons, triglycerides com­ prise 70-80%, phospholipids 15-20%, and cholesterol esters are present in small amounts (Felinsky et al., 1964), About one-third of chylomicrons should be absorbed by the adipose tissue, one-third by liver parenchymal cells (Di Luzio, 1960), the rest by other tissues (Robinson, 1963). The importance of blood lipid composition, is also apparent for milk fats, if we consider that 5 0 % of milk lipids derive from blood (especially long-chain fatty acids) (Riis et al., 1960) and the rest is synthesized by the mammary gland (short-chain fatty acids). Of blood lipids, chylomicrons and low density lipoproteins are utilized (Barry et al, 1963). The 142 fatty acids identified in milk can be classified in the fol­ lowing way. Saturated fatty acids from 2 to 28 carbons represent 72-83%; monoenic acids from 10 to 26 carbons, with positional and structural isomers (mostly of C 18 compounds) are 30.75%; dienoic

4.5 1.9

25.9 23.6

27.1

23

27 30 26.5 35.9 24

C16:0

31

0.2

C14:l

5

5 4 3.9 5.1 3

C14:0

"Mattson efaZ. (1964). "Hilditch and Longenecker (1937). c Shorland(1955). "Barker and Hilditch (1950). e Gupta and Hilditch (1951). Gutter and Shorland (1957).

Horse'' Rabbit

Ruminants Sheep" Beef Ox" Deer c White-tailed deer" Pseudo-ruminants Camel" Ruminantlike Hippopotamus rf

Species

Table XVIII

22.2

2.2 4.7 4.9

31

4

6.8 5.2

27 25 23.1 29.6 36

C18:0

Fatty acids

3 5 5.1 2.2 3

C16:l

1.5 16.2 39.7

5.2 8.9 33.7 12.7

Traces

1 0 0 7.7

C18:3

3.5

1

2 1 1.8 1.2 1

C18:2

39.3

28

35 36 40.4 17.0 2

C18:l

F A T T Y A C I D C O M P O S I T I O N ( W E I G H T P E R C E N T ) O F D E P O T F A T S O F S E V E R A L H E R B I V O R O U S ANIMALS

2.5 2.3

1.4

1.2

Others

2

> o <

2 O

0.47 0.11 0.30

"Keeneyefa/. (1962a). "Garton (1960). c Kelsey and Longenecker (1941). "Viviani etal. (1963c).

Esterified fatty acids Phospholipids

FFA

Glycerides plus FFA Glycerides Cholesterol ester

Lipid Fractions

Table XIX

0.20

C13:0 br

0.90

0.20 0.83

C14:0 br

0.60

0.20 0.28 Traces

0.81 0.40 0.60

0.06 0.20 0.40

0.60

1.30

1.70

C16:0 br

C15:0 br 23.50 2.40 4.80 5.50 11.10 18.50 34.40 15.43 16.61 20.90 16.20

C16:0

1.02 1.07 2.20

4.20

5.30

C16:l

3.70

C17:0 br

0.35 0.42

_ -

-

_ _ _

C17:l

3.27 2.33

-

C17:0 17.40 30.00 0.50 1.50 3.30 24.10 5.20 30.67 17.34 21.10 26.80

C18:0

-

19.95 28.94 29.10 14.10

-

17.61 13.86 15.60 16.30

18.10 4.60 80.50 52.40 61.70 5.20 27.30 24.30 3.30 5.60 7.90 39.30

C18:2

SERUM

C18:l

FATTY ACID COMPOSITION ( W E I G H T PERCENT) O F L I P I D FRACTIONS FROM BOVINE B L O O D

-

-

2.40

0.47 1.08

-

C20:3

6.65 11.97

-

-

1.90 20.00 22.90 9.20 3.30

C18:3

-

3.13 3.79

-

C20:4

328

ROMANO VIVIANI Table XX

FATTY ACID COMPOSITION (WEIGHT PERCENT) O F D I F F E R E N T

PHOSPHOLIPIDS

F R O M VARIOUS O X T I S S U E S "

Liver Fatty acid

Lecithin

C 10:0 C 11:0 C 12:0 br C 12:0 C 13:0 C 14:0 C 15:0 br C 15:0 br C 15:0 C 16:0 br C 16:0 C 16:1 C 17:0 br C 17:0 C 17:1 C 18:0 br C 18:0 C 18:1 C 18:1 iso C 18:2 C 18:3 Unknown C 20:3 C 20:4 C20:5 Unknown Unknown C 22:5 C 22:6



Traces Traces



Traces

— 0.20 0.20

-

12.70 0.60 0.60 0.70

— —

Heart

Cephalin

— —

0.10



Traces 0.10 0.20 0.10 0.10 12.30 0.30 1.00 1.20

— —

35.00 14.30 1.60 7.70 Traces

39.40 13.20 6.50 6.80

-

Traces 1.30 11.10

3.50 7.80 2.60 0.60 1.20 9.60 1.50

— — 5.10 1.10

Lecithin

— — _ —

0.40

— — 0.50 0.40 36.70 4.20 1.40 1.00 1.50 Traces 8.70 30.00

— 14.50 1.30

— — — — — — —

Spleen

Cephalin Traces Traces Traces Traces



0.20

— Traces Traces Traces 3.50 1.20 0.80 1.00

Lecithin

Cephalin

-

— —

Traces



Traces



0.20



— — — —

0.30 1.20 1.00 45.00 1.60 3.30 1.40

— —

58.00 6.50 2.90 13.60 0.90

16.30 19.10

Traces 11.60 Traces

_ — — _



0.60

— —

-

0.20

5.80 0.40

_ 0.60 0.60

— 63.00 21.40





6.10

4.30

— —

— —

0.90 3.50

_ _ _ — _

Traces 6.90 Traces

— — — —

"Gray (1960).

acids from 14 to 26 carbons are 2.97%; and polyenoic acids (exclu­ sively 18 to 22 carbons compounds) are only 0.85%; monobranched acids of 16 to 28 carbons are 0.82%. Fatty acids of different nature, such as keto or hydroxy compounds are 0.40% (Jensen et al., 1967). Thus, in addition to geometrical and positional isomers of C 18:1, C 18:2, and C 18:3, trace amount of /3-ketoacids from C 6 to C 16 (Van der Ven et al., 1963) and also other ketoacids such as ketopalmitic and

Metabolism

of Long-Chain

Fatty Acids in the Rumen

329

ketostearic acids (Keeney et al, 1962b), a compound having 4 methyl groups, in positions 3, 7, 11, and 15, also has b e e n demonstrated in milk (Sonneveld et a/., 1962). Depot fats of adult ruminants, as a consequence of the activity of rumen microorganism, are "stearic rich," and this peculiar character­ istic allows a clear distinction from depot fat of other herbivorous, nonruminant animals (Table XVIII). Also tissue lipids can be influenced by rumen fatty acid content (Tables XIX and XX). Duncan and Garton (1963) reported that in one animal the multibranched acids represented 4 3 % of total fatty acids of blood phospholipids. In a plasmalogen fraction of ram spleen, liver, and sperm, it has b e e n observed that 5 0 % of the fatty aldehydes are branched (Gray, 1960). Hawke (1956) found that, together with longchain fatty acids, ox liver phospholipids contained traces of all lower fatty acids, from formic to caproic acid, and also isobutyrate, isovalerate, and /3-methylbutyrate. VII.

Effect of Rumen Metabolism on the Fatty Acid Composition of Ruminant Lipids

A. EFFECTS OF R U M E N FUNCTION DEVELOPMENT Young ruminants fed a milk diet behave like monogastric animals: i.e., they do not modify in their digestive apparatus the chemical na­ ture of fatty acids, and moreover they have an EFA nutritional re­ quirement (Cunningham and Loosli, 1954; Lambert et al., 1953; Falaschini et al., 1965). In the change from a milk diet to a mixed diet of hay and concentrates, the animal becomes a ruminant: the ruminal micropopulation takes its place, and, accordingly, volatile fatty acids increase and glucose decreases in blood, ruminal papillae develop, and the mobility of prestomachs is activated. In such conditions, the EFA requirement disappears and dietary fatty acids begin to undergo substantial modification. During the development of the ruminal function, it has b e e n ob­ served that a diet of concentrates in comparison with a diet of hay plus concentrates increases the total fatty acid content (Table XXI) and effects more slowly the installation of a bacterial population capable of biohydrogenation (Viviani et al, 1967e) (Table XXII). Considering the higher lipid content of the digesta of calves fed concentrates, the

Table XXI

1.16 1.17 1.11 1.21

Ration 1, luzerne plus concentrates^ 2.24 3.21 3.10 3.13

± 0.45 ± 0.95 ±0.59 ±0.68

Rumen fluid of calf fed ration 1 (4)c

"Vivitmietal. (1967e). ''Values are expressed as milligrams per gram of dry material. c The numbers in parentheses are the number of animals.

1-15 16-29 30-57 58-71

Length of period (days) 1.11 1.11 1.11 1.11

Ration 2, concentrates

4.64 5.80 7.35 5.55

± ± ± ±

0.89 0.83 0.97 0.50

Rumen fluid of calf fed ration 2 (4)

p p p p

< < < <

0.01 0.05 0.01 0.01

Significant difference

E F F E C T O F T W O D I F F E R E N T RATIONS O N T O T A L L O N G - C H A I N F A T T Y A C I D C O N T E N T IN D R Y R U M E N F L U I D O F C A L F "

ROMANO VIVIANI

C C C C C C C C C C C C C C C C C C

0.88 0.16 2.11 9.64 33.28 18.82



0.46 0.43 24.15 4.38



0.08 0.28 1.55 0.68 2.41 0.33 23.04 2.59 1.05 1.30 0.90 23.12 20.03 14.88 2.25

0.51 0.44 2.03



0.03 0.04

0.14 0.58



Sample at day 15

0.05 0.37 0.04 0.20 0.47 1.38 2.71 1.97 0.67 21.14 1.14 0.73 0.71 0.57 46.44 10.96 4.59 2.25

Sample at day 29

Rumen fluid of calf fed ration 1

F U N C T I O N IN T H E



0.45 0.09 0.37 13.17 57.58 6.21



0.15 0.03 14.98 2.93

1.08

— — —

0.03 0.08

Ration 2, concentrates

CALF0-6

0.02 0.36 0.02 0.06 0.32 0.75 2.86 0.94 0.46 16.58 1.32 1.17 0.60 0.26 14.15 38.94 14.69 1.49

0.06 0.44 0.10 0.16 0.33 1.00 2.83 0.98 1.07 19.14 1.39 0.67 1.12 0.05 42.00 14.20 10.62 1.29

Sample at day 29

Rumen fluid of calf fed ration 2 Sample at day 15

of Long-Chain

"Viviani et al. (1967e). 6 Values are expressed as weight percent calculated from peak areas.

10:0 12:0 13:0 br 13:0 14:0 br 14:0 15:0 br 15:0 16:0 br 16:0 16:1 17:0 br 17:0 18:0 br 18:0 18:1 18:2 18:3

Fatty acid

Table XXII

D I F F E R E N T R A T I O N S O N L O N G - C H A I N F A T T Y A C I D C O M P O S I T I O N DURING T H E D E V E L O P M E N T O F R U M I N A L

Ration 1, luzerne plus concentrates

E F F E C T OF Two

Metabolism Go 03

Fatty Acids in the Rumen

Table XXIII

br

br

br

br



br

0.064 0.011 0.120 1.540 3.389 3.559 0.014



0.012 0.009 1.677 0.462



0.012 0.010 0.085

br

0.011" 0.039

0.003 d 0.009

+ 0.031 c + 0.177 + 0.035 + 0.030 + 0.212 + 0.510 + 0.648 + 0.432 + 0.141 + 4.611 + 0.038 + 0.124 + 0.142 + 0.244 +16.222 + 1.970 - 2.209 - 2.702 + 0.263

0.042 d 0.246 0.035 0.042 0.222 0.595 0.648 0.444 0.150 6.288 0.500 0.124 0.206 0.255 16.342 3.510 1.180 0.857 0.277



0.051 0.010 0.041 1.467 6.414 0.691 0.105

0.017 0.003 1.669 0.326



0.120

— — —

Ration 2, concentrates

Deviation from ration 1

Rumen fluid of calf fed ration 1

Sources of long-chain fatty acids

TREATMENT0-6

-

0.064 d 0.437 0.084 0.071 0.069 0.789 1.326 0.727 0.242 10.181 0.936 0.135 0.227 0.007 25.360 9.301 4.406 0.336

Rumen fluid of calf fed ration 2

+ 0.06F + 0.434 + 0.084 + 0.071 + 0.069 + 0.669 + 1.326 + 0.710 + 0.239 + 8.572 + 0.610 + 0.135 + 0.176 - 0.003 +25.319 + 7.834 - 2.009 - 0.355 - 0.105

Deviation from ration 2

71

"Vivi&nietal (1967e). 6 Values are expressed as milligrams per gram of dry material. C a l c u l a t e d by difference; figures indicate the changes of individual fatty acids of rumen fluid in comparison to the diet. T h e signs + and — indicate respectively an increase or a decrease of the individual fatty acid content in rumen fluid in comparison to the diet. "Values were derived from the percentage of the individual fatty acids times the total amount of long-chain fatty acid per gram of dry material (rumen fluid and diet) divided by 100.

C 10:0 C 12:0 C 13:0 C 13:0 C 14:0 C 14:0 C 15:0 C 15:0 C 16:0 C 16:0 C 16:1 C 17:0 C 17:0 C 18:0 C 18:0 C 18:1 C 18:2 C 18:3 Others

Fatty acid

Ration 1, luzerne plus concentrates

DAYS O F

E F F E C T O F T W O D I F F E R E N T R A T I O N S O N T H E I N D I V I D U A L F A T T Y A C I D C O N T E N T IN D R Y R U M E N F L U I D O F C A L F A F T E R

2

>

> o <

O

to

00 00

Metabolism

of Long-Chain

Fatty Acids in the Rumen

333

dietary condition appears to modify the type of energy supplied to the animal in the period of the development of ruminal function. The metabolic process involving the biohydrogenation of unsaturated fatty acids begins at least after 15 days of nonmilk diet. It appears that a diet with high roughage is most efficient for producing microorganisms active in enzymatic biohydrogenation. The same percentage in rumen digesta of branched C 15:0 after 15 days on either diet indicates that in the microbial colonization of the rumen the most typical microorga­ nisms are those producing and containing C 15:0 branched acids. After one month the fatty acid spectrum on either diet resembles that of adult ruminants. Also the total fatty acid content, which de­ pends upon the diet type, is the same as in the adult animal. After one month of a hay or concentrates diet, not only the amount of volatile acids (Barnett and Reid, 1961, p. 94), but also the type of long-chain fatty acids, is similar to that of animals with developed rumen func­ tion (Table XXIII) (Sections III and IV). Masters (1964) has also studied the development of rumen in rela­ tion to the modifications of tissue fatty acids: he found a progressive increase of stearic acid and a decrease of oleic acid both in fat depots and in the neutral fat fractions of heart, kidney, and liver. B. E F F E C T S O F D I E T A R Y

CONDITIONS

A characteristic distinguishing ruminants from other non-ruminant herbivorous (and in general all monogastric) animals, is the relative insensitivity of their fats to the influence of dietary lipids: the extent of unsaturation in tissues does not d e p e n d on the unsaturation of dietary lipid (Thomas et al, 1934). T h e role of rumen is clear on depot fats after administration either intravenously or by duodenal cannulation, of fatty acids, and also during fasting. All experiments that involve bypass of the rumen bring about an increase of linoleic and linolenic acids in depot fats (Ogilvie et al.9 1961; Tove and Mochrie, 1963). In a prolonged fast of 7 days there is in bovines a clear decrease of stearic acid and an increase of oleic and palmitic acids in blood lipids in comparison with the fed animals. The considerable fall of stearic acid is a direct consequence of the lack of the ruminal contribution to the fatty acid pool (Viviani et al., 1963c). Nevertheless some alterations can be observed even in ruminants, depending on diet changes. According to McCarthy (1962a), analysis of milk fat from a cow re­ ceiving a low roughage and high concentrate ration results in a de­ crease in the fat content of milk, but in increase of the percentage of

334

ROMANO VIVIANI

linoleic acid to 8.6% and of linolenic to 2 . 3 % of total fatty acids. These levels are not usually found in ruminant milk. By using purified diets, higher levels of oleic acid are observed until depot fats are liquid (Tove and Matrone, 1962). An increase of the cereals in the diet of bovines induces an increase of unsaturated acids and a decrease of the saturated ones. On the con­ trary the increase of hay causes opposite effects (Kunsman and Keeney, 1963). Pelleting, in comparison with the same rough diet, has induced an increase of iodine number in depot fats, associated with a marked modification of volatile fatty acids in rumen (Shaw et al., 1960). It is conceivable that these phenomena are due not only to the chemical composition of the diets (i.e., starch and linoleic acid on one hand, cellulose and linolenic acid on the other hand), but also to the physical state of the diets (rough or powdered) which modifies the ruminal ecology. These results may be explained from the experi­ ments which consider the effect of dietary conditions on long-chain fatty acid content in the rumen. A larger quantity (on the basis of dry weight of rumen fluid digesta) of long-chain fatty acids (also polyunsaturated fatty acids and branched fatty acids) was present in calves fed a high concentrate ration, in comparison with calves fed high roughage, after 71 days of treatment (Viviani et al., 1967e) (Table XXIII). Since rumen biohydrogenation could be considered the main con­ trol mechanism for unsaturated fatty acids of the ruminant body, our data indicate that all concentrate diets induce higher levels of unsatu­ rated fatty acids primarily in the rumen, and later in the body fats. Increasing the dietary lipids brings about an increase of stearic acid in depot fats (Edwards et al., 1961), and of oleic acid, in addition to stearic acid, in milk lipids (Brown et al., 1962). Such a difference in lipogenetic behavior between adipose tissue and the mammary gland was particularly stressed in the investigations of Tove and Mochrie (1963); these authors pointed out that the increase of stearic and oleic acids in milk is accompanied by a decrease of palmitic and myristic acids, while the increase of stearic acid in adipose tissues is concomi­ tant with the decrease of oleic acid. Lauryssens et al. (1961) made a study with labeled stearic acid and concluded that in the mammary gland there is a higher capability of transformation of stearic to oleic acid in comparison with the adipose tissues. According to Reiser et al. (1963) the high levels of stearic acid in ruminant tissues would d e p e n d not only upon the high absorption of stearic acid, derived from biohydrogenation in the rumen, but also on

Metabolism

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Fatty Acids in the Rumen

335

the presence of substances formed in the rumen itself, which are re­ sponsible for inhibition of the transformation of stearic to oleic acid in the tissues. This hypothesis is derived from the fact that cottonseed oil (containing sterculic acid) (Section II) induces in pigs a more solid fat and enhances the levels of stearic acid (Harrington and Adriance, 1893). Since, according to Reiser et al. (1963), in rumen there is lactobacillic acid (which resembles sterculic acid), this acid might also in­ hibit the reaction of stearic to oleic acid. Investigations in yeast and rats have demonstrated, however, that lactobacillic acid does not in­ hibit the conversion of C 18:0 to C 18:1. Subsequent experiments have indicated that only cyclopropene acids inhibit fatty acid desaturase (Raju and Reiser, 1967). Since this acid is found only in certain plants, but not in rumen bacteria, the problem is still open. Finally a particular interest, both biochemical and technological, is in the variation of trans fatty acids in butter and depot fats. It is known that the content of these acids depends on the types of polyunsaturated acids present in dietary lipids and on their biohydrogenation in the rumen. Thus, it was demonstrated that intake of linolenic acid (in the form of linseed oil) by milk-producing cows, enhances the content of trans acids, of stearic acid, of conjugated, di- and trienoic acids in milk, while C 14:0, C 16:0, and odd and branched acids decrease (Kunzdzal-Savoie et al., 1966). In general a pasture alimentation in­ duces an increase of trans acids, of oleic and stearic acids, and a de­ crease of the other saturated acids: this is a consequence of the ele­ vated levels of linolenic acid in the lipids of fresh hay. Feeding concentrates, which are richer in linoleic acid, induces in milk lower levels of trans acids, more linoleic acid, less saturated acids. There is therefore a "winter" pattern and a "summer" pattern for milk. Kaufmann et al. (1961) have demonstrated that 6.9% of total fatty acids are trans in summer milk, whereas trans acids are less than 2 % in winter milk; they are less than 0.4% in human milk. Such differences might be very interesting both for technological and nutritional purposes. These observations suggest also the problem of using concentrates whose fats are obtained by hydrogenated fats having trans fatty acids: these concentrates could bring the levels of trans acids to those in the milk of animals at pasture, or even higher. In this connection, w e should keep in mind the proposal by Bartlet and Chapman (1961) to use, as a means of revealing the addition of hydrogenated fats to butter, the ratio of infrared absorption bands of conjugated cis-trans unsaturated acids to those of trans isolated acids. In addition, Strocchi et al. (1967) observed a correlation of conju­ gated dienoic acids of milk with trans isomers and linoleic acid in the

336

ROMANO VIVIANI

diet. In the Emilian region of Italy, in areas where dietary conditions are different, these patterns change in summer butter in comparison with winter butter; on the other hand, in areas were diets are more constant for the production of "Parmigiano-Reggiano" cheese, such patterns are the same throughout the year. Unsaturated fatty acids of milk are also important in the chemistry of flavors. Begemann and Koster reported (1964) the presence in milk fat of the important flavoring compound 4-cis-heptanol and showed that it originated through the autoxidation of one or more of the fatty acids. Based upon the classical autoxidation theory, D e Jong and Van der Wei (1964) proposed the A ii, is o r Aio,i5 octadecadienoic acids as pos­ sible precursors, and reported the presence of both these fatty acids in milk fat. These fatty acids, and therefore flavor, could d e p e n d on hydrogenation processes in the rumen. VIII.

Long-Chain Fatty Acids in Rumen Dysfunction

Ruminants in contrast to other animals, present certain particular pathological patterns that d e p e n d on rumen. Fatty acids appear to be involved in ruminant pathology, both at the level of rumen dysfunc­ tion and in other tissues. Current knowledge concerns chiefly bloat; it is possible, however, that also muscular dystrophy may be related to rumen metabolism. Numerous experiments furnish evidence that both in muscular dys­ trophy produced experimentally (Baxter et aL, 1952; Adams et al., 1954) and in enzootic forms of the disease (Poukka, 1966), unsaturated fatty acids may play an important role. The factors that make the tis­ sues accumulate unsaturated fatty acids are not known. It is possible that in young calves the rapid increase of polyunsaturated fatty acids together with deficiency of vitamin E and selenium are important fac­ tors in inducing muscular dystrophy. Thus the unsaturated fatty acid levels (which could d e p e n d on fatty acid metabolism in the rumen) of milk fat of cows, in areas where this disease occurs enzootically, may be important if selenium and vitamin E are deficient. Data are lacking, however, on this subject. A.

BLOAT

A characteristic disease of ruminants which depends upon ab­ normal fermentation in rumen is bloat. There are two types of bloat: frothy bloat and free gas bloat (Hungate, 1966, p. 442). In both, a build-up of pressure in the rumen and an inability to eructate the fer-

Metabolism

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Fatty Acids in the Rumen

337

mentation gases is present. Among the various types of bloat, the one deriving from legumes is always of the frothy type; a different form recently discovered is feedlot bloat, which at times can produce also free gas bloat. Both for legume bloat and for the other type, lipids and fatty acids appear to be involved. The prophylactic and therapeutic use of vege­ table oil, emulsified tallow, whale oil, or cream as antifoaming agents in treating the bloat in cows fed on red clover, has b e e n reported by New Zealand workers (Johns, 1954; Reid, 1955). Reid (1956,1958) has developed a technique of spraying dangerous pastures with the emul­ sified oil. Antibiotics are also used for control of bloat. The administra­ tion of penicillin can prevent legume bloat (Chivers, 1954; Barrentine et al., 1956). It was tentatively suggested that part of this effect may be due to a decrease in bacterial breakdown of plant lipids, which are considered to act as antifoaming agents in vivo (Wright, 1961). The lipid foam-breaking theory indicates that the stability of the froth is less in the presence of alfalfa chloroplasts. When lipids were re­ moved, the chloroplasts were not effective in breaking the froth (Mangan, 1959). The effectiveness of lipids in breaking the froth appeared to be diminished as the lipid was hydrogenated. The rate of release of galactolipid from forage varied, but did not correlate with bloat (Bai­ ley, 1964). Chemical analyses show, in the material surrounding the bubbles, a high percentage of lipid, carotenoid pigment, and nucleic acids (Bartley and Bassette, 1961). The slime does not contain lipids (Gutierrez et al, 1963). With intensive feeding of a ration high in grain, a feedlot bloat oc­ curs (Smith et al., 1953). Since the occurrence of this form of bloat is of recent observation, thus also its economic importance and the way to fight it are not well known. In the legume bloat, lipids are of beneficial effect, whereas in feedlot bloat they are dangerous. In animals with feedlot bloat the disease is intensified by the addition of 4 - 8 % soy­ bean oil (Emery et ah, 1960). B.

PARAKERATOSIS

A disease associated with feedlot bloat is parakeratosis. A disorder involving the rumen wall occurs w h e n finely ground roughage is pelteted and fed with concentrates (Beardsley, 1964). This condition, first observed in lambs on pelleted feed containing alfalfa, is ruminal parakeratosis, a condition characterized grossly by hardening, enlargement, and clumping of mucosal papillae (Fig. 9)

A

B

F I G . 9 Ruminal parakeratosis. Comparison of the inner surfaces of two calf rumens; the first calf (A) was fed forages, the second one (B) was fed only concentrates. In the second calf, the hypertrophic appearance and the darker color of the inner mucosa are quite evident (Montroni et al., 1968).

Metabolism

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Fatty Acids in the Rumen

339

and microscopically by accumulation of excessive layers of keratinized, nucleated, squamous epithelial cells of papillae (Jensen et al., 1958). This disease may be associated with liver abscesses (Montroni et al., 1968). Hyperplasia of papillae is perhaps also linked to volatile fatty acids, which have b e e n shown to stimulate mRNA biosynthesis (Viviani et al, 1967a). Parakeratosis was more severe in animals fed pelleted rations and was aggravated by high concentrate addition. T h e gravity of parakera­ tosis was decreased by the addition of 12% sodium bicarbonate (Meyer et al, 1965). Feeding 3 % bicarbonate in the ration can increase the acetate to propionate ratio and maintain the milk fat content when high grain rations are fed (Davis et al., 1964; Emery et al., 1964). In animals there is, according to studies of Meyer et al. (1965), an increase of body fat with the aggravation of the disease. Such increase of body fat may be accompanied by increase of lipid content in rumen. This observa­ tion can also be related to our findings (Viviani et al., 1967e) that in calves fed concentrates there is a 2- to 3-fold increase of the fatty acid content in comparison to that of animals fed hay plus concentrates (the lipid content of the diets being the same) (Tables XXI and XXIII). It is possible that, in rumen of calves fed concentrates, the increase of long-chain fatty acids may be the consequence either of an in­ creased production of volatile fatty acids or of their decreased absorp­ tion and their utilization for biosynthesis of higher fatty acids. In fact, in such conditions other investigators observed high levels of volatile fatty acids (Balch, 1960; Moore, 1964). Our studies have shown that in calves fed concentrates, in which parakeratosis occurs, also high quan­ tities of long-chain fatty acids are present in comparison with animals fed hay and having a normal ruminal mucosa (Viviani et al., 1968b). On the other hand, an increased breakdown of carbohydrate or protein to gases, or an increased absorption of their metabolites could also determine a relative increase of long-chain fatty acids. Furthermore, in the rumen of animals fed concentrates, stearic acid and branched fatty acids are present in lower levels than in those fed lucerne hay. IX.

Conclusions

T h e studies that we have reviewed reveal the importance of veteri­ nary problems in stimulating new interest in biochemical processes that might improve foods of animal origin and, thus, human nutrition. Furthermore, the investigations reported above have contributed to

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one of the most interesting areas of biochemical studies: the molec­ ular basis of evolution. They have allowed the discovery of living bio­ chemical fossils and have helped to elucidate the mechanisms of branched fatty acid biosynthesis and unsaturated fatty acid biohydrogenation. Branched fatty acids are now of primary interest because it is hoped to understand through their chemical language the complete phe­ nomena of evolution since the origin of life. Studies on the origin of petroleum in association with studies on biological evolution show that branched-chain hydrocarbon structures derive from the corre­ sponding fatty acid molecules. Analysis of sediments of 2.7 billions of years ago has revealed the presence of the isoprenoid alkanes pristane and phytane. This is extremely interesting since, if such isoprene compounds are derived from phytol of chlorophyll, a more pre­ cise date can be given for the beginning of life on earth (Eglinton and Calvin, 1967). In fatty acid biohydrogenation, the enzyme responsible for the dis­ appearance of a 77 bond probably represents a vestige (whose deep biological meaning is now hidden in the rumen) of the mechanisms of energy transformations of primitive organisms, w h e n there was no oxygen in the atmosphere, at the beginning of life on earth. References Ackman, R. G., and Hooper, S. N. (1968). Comp. Biochem. Physiol. 24, 549. Adams, E. P., and Heath, T. J. (1963). Biochim. Biophys. Ada 70, 688. Adams, R. S., Gullickson, T. W., Sautter, J. H., and Gander, J. E. (1954). J. Dairy Sci. 37, 655. Allison, M. J. (1965). In "Physiology of Digestion in the Ruminant" (R. W. Dougherty et al, eds.), p. 369. Butterworths, London. Allison, M. J., Bryant, M. P., Katz, I., and Keeney, M. (1962) J . Bacteriol. 83, 1084. Annison, E. F., and Lewis, D. (1959). In "Metabolism in the R u m e n " (R. Peters and F. G. Young, eds.). Methuen, London. Bailey, R. W. (1964). New Zealand J. Agr. Bes. 7, 417. Balch, C. C. (1960). Proc. 8th Int. Grassland Congr. Beading, EngL, p. 528, Grassland Res. I n s t , Hurley, England. Baldwin, R. I. (1965). In "Physiology of Digestion in the Ruminant" (R. W. Dougherty et al, eds.), p. 379. Butterworths, London. Banks, A., and Hilditch, T. P. (1931). Biochem. J. 25,1168. Barker, C., and Hilditch, T. P. (1950) J . Chem. Soc. p. 3141. Barnett, A. J. G., and Reid, R. D. (1961). "Reaction in the Rumen." Arnold, London. Barrentine, B. F., Shawver, C. B., and Williams, L. W. (1956) J . Animal Sci. 15, 440. Barry, J. M., Bartley, W., Cinzell, J. L., and Robinson, D. S. (1963). Biochem. J. 89, 6. Bartlet, J. C., and Chapman, D. G. (1961) J . Agr. Food Chem. 9, 50. Bartley, E. E., and Bassette, R. (1961) J . Dairy Sci. 44, 1365. Bauchop, T., and Elsden, S. R. (1960). J. Gen. Microbiol. 23, 457.

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