The effects of diet and other factors on the lipid composition of ruminant tissues and milk

0079-6832 7q 0101-0245S05.(X) 0

Pro#. Lipid Res. Vol, 17. pp. 245 277. © Pergamon Press Lid. 1979. Printed in Great Britain

THE LIPID

EFFECTS

OF

COMPOSITION

DIET OF

AND

OTHER

RUMINANT

FACTORS TISSUES

ON AND

THE MILK

W . W . CHRISTIE

Biochemistry Department, The Hannah Research Institute, Ayr, Scotland KA6 5HL CONTENTS I,

II.

INTRODUCTION

245

DIET AND O T H E R FACTORS AND PLASMA LIPID COMPOSITION

247 247 248 249 254 254

A. B. C. D. E.

Plasma lipid composition in young and developing ruminants Plasma lipid composition during pregnancy and lactation Dietary fatty acids and plasma lipid composition Heat and cold exposure and plasma lipid composition Plasma lipid changes during fasting

III. DIET AND OTHER FACTORS AND THE LIPID COMPOSITIONOF ADIPOSE TISSUE AND SKELETAL MUSCLE A. Lipid composition in adipose tissue and skeletal muscle of young and developing ruminants B. Dietary fatty acids and adipose tissue and skeletal muscle composition in adult ruminants C. Branched-chain fatty acids in adipose tissue

255 255 256 260

IV. DIET AND OTHER FACTORS AND THE YIELD AND COMPOSITIONOF MILK FAT A. The effect of breed, stage of lactation and season on milk fat composition

261 261 262 262 263 268 270 271

B. Dietary fatty acids and the yield and composition of milk fat I. Some general considerations 2. Saturated and monoenoic fatty acids 3. Polyunsaturated fatty acids of vegetable oils 4. Oligounsaturated fatty acids of cod-liver oil C. The low milk fat syndrome V. REFERENCES

272

I. I N T R O D U C T I O N

Fatty acids in the tissues of ruminant animals have their origin in a variety of sources. Many of the dietary long-chain saturated fatty acids pass through the rumen unchanged and they are subsequently absorbed and incorporated into the animal tissues but dietary unsaturated fatty acids are subjected to hydrogenation or partial hydrogenation by rumen microorganisms before passing into the intestinal tract. Fatty acids synthesized de novo by rumen microorganisms are eventually released and are taken up by the animal as the microorganisms themselves are digested. Within the animal itself, fatty acids are formed by synthesis de novo from short-chain precursors or by modification of other dietary components, e.g. oxidation of phytol to phytanic acid and by ~- or B-oxidation, desaturation or chain elongation of dietary fatty acids. These various processes are discussed in some detail elsewhere 111b't73~.2S8" and all are susceptible to some extent to dietary modification. As an example, dietary linoleic acid (9c,12c-18:2) is hydrogenated partially in the rumen yielding stearic acid (18:0), vaccenic acid (11t-18:1) and isomers and a conjugated dienoic acid (9c,11t-18:2). All of these fatty acids are absorbed and re-esterified in the integtines and ultimately are circulated to all parts of the animal. Some unchanged linoleic acid may then be converted to arachidonic acid and other longer-chain fatty acids, while the stearic acid may be desaturated to oleic acid (9c-18:1). In addition, all may be partially oxidized, 11t-18:1 yielding small amounts of 10t-17:1 and 9t-16:1 fatty acids, for example. Supplementation of the diet of ruminant animals with linoleic acid, therefore, may not produce any elevation of the concentration of this component in the tissues but the levels of 18:0 or 18:1 fatty acids may increase. Desaturation of stearic acid to oleic acid is a particularly active process in ruminant animals and takes place at many sites within the animal including the intestines, adipose J.P.L.R. 17 3---A

245

246

w.w. Christie

tissue and mammary gland. The feeding of dietary supplements of stearic acid land most other Cls fatty acids) to ruminant animals, therefore, leads to the synthesis of fats rich in oleic acid and these may have comparatively low melting or softening points. On the other hand, dietary supplements of palmitic acid are not metabolized further to any degree by the animal and palmitic acid is deposited as such giving fats that have comparatively high melting points. Dietary short-chain fatty acids (C12 and below) may be absorbed from the intestinal tract in a different way from longer-chain components (see Noble 173~') but generally are chain-elongated before deposition in the tissues. For example, dietary supplements of lauric acid can give rise to elevated tissue levels of myristic and palmitic acids. The typical diet of ruminant animals consists of fresh or conserved herbage and other plant products such as cereals. These materials do not, in general, contain large amounts of lipid and that present tends to have linoleic and linolenic acids as the main fatty acid constituents (see Harfoot 111h and Noblel~3"). Under normal conditions. high proportions of these fatty acids are hydrogenated in the tureen before they reach the intestines. The lipid compositions of the tissues of ruminant animals on such normal or control diets were discussed in a previous article. 39a It is, however, feasible to vat5 the fat composition and content of ruminant diets by supplementation with fats or oils of known composition. Apart from intellectual curiosity, there are sound commercial reasons for attempting this. For example, there have been attempts to increase thu energy intake of the animals by this means and so stimulate fat, especially milk tat. production and to convert cheap fatty by-products of the food industry such as tallow to a more commercially valuable form such as butter. Also, in view of current concern over possible relationships between the content of saturated fatty acids in the human diet and atherosclerosis, there is a considerable degree of interest in increasing the polyunsaturated fatty acid content of those ruminant fats that are ultimately destined for human consumption. Dietary fat supplements are normally subjected to the same processes within the' ruminant animal as the fat in the basal ration, i.e. they are hydrolyzed rapidly in the tureen and unsaturated components are hydrogenated before passing e~entuall? to flw intestines and hence, into the animal tissues. On the other hand, elevated concentrations of free (unesterified) fatty acids can affect the metabolic activity of rumen microorganisms and so alter such processes as cellulose or protein breakdown or volatile fatty acid (VFA) production. Similarly, high concentrations of free fatty acids may have an effect on the absorption process and thence, on digestibility of fats, possibly with suppression of appetite. Once absorbed into the animal tissues, dietary fatty acids may modify lipid metabolism and the lipid composition of tissues by inhibiting or stimulating farts acid synthesis or desaturation, for example. The complexity of these processes is such thai it is not always possible to predict the effect of supplementing the diet of a ruminant animal with specific fats on the ultimate composition of the tissue lipids of the animal. The effects of dietary fats on rumen microorganisms, or vice versa, can be eliminatcd or avoided by a number of experimental procedures. New-born ruminants, to all intents and purposes, do not possess a rumen and the effects of dietary fatty acids on the compositions of tissue lipids of sheep and calves in the first few weeks of life are discussed elsewhere.173b The development of rumen function in young ruminants can bc delayed by continued feeding with liquids thereby stimulating closure of the oesophageal groove and allowing the liquid diets to pass directly into the abomasum (see Harfoot ~ " ) so that any unsaturated fatty acids present in the milk or milk substitute escape biohydrogenation. ~9'*.2ss'3°1 With adult ruminants, the rumen and its microorganisms have been by-passed by infusing fat suspensions directly into the abomasum or duodenum after modifying the animals by appropriate surgical techniques ~84 or indeed directly into the bloodstream via the jugular vein. 2s2 The latter procedures, though useful for experimental purposes with small numbers of animals or for brief time periods, have little practical value. Methods of preparing diets for ruminant animals that contain fats and oils in a form that provides protection against hydrolysis and biohydrogenation

Effects of diet on lipid composition

247

in the rumen have been developed, however. Initially, a6'225'226 this was achieved by encapsulating minute oil droplets in formaldehyde-treated casein which was resistant to proteolysis within the rumen but was hydrolyzed in the acidic conditions of the abomasum. The oil droplets were protected from microbial action during passage through the rumen but were liberated in the abomasum and became available for hydrolysis and absorption in the small intestine. Later it proved equally effective but more economical to use formaldehyde-treated oil seeds in a similar way. 223 An alternative related method in which the protein encapsulating the oil is protected from microbial action by a tanning procedure has been described. 13a'3°5 The uses of protected lipids in ruminant nutrition have been reviewed. 106,224.253 In addition to fat supplementation of the diet, the lipid composition of the tissues of ruminants may be affected by a number of other factors including the content and composition of other dietary components (e.g. roughage), pregnancy, lactation, season and climate. Also, the nature and biological activity of the rumen microflora can vary considerably between herds or even between animals in the same herd and are also subject to many outside influences in ways that are not readily quantifiable. The fatty acids present in any tissue will consist of fatty acids synthesized de novo in the tissue and those synthesized elsewhere in the animal or obtained from the diet and transported to the tissue in the plasma. The manner in which the plasma lipids change in response to any of the factors listed above will, therefore, have an important bearing on the composition of the other tissue lipids. II. D I E T A N D O T H E R

FACTORS

AND PLASMA LIPID COMPOSITION

A. Plasma Lipid Composition in Young and Developing Ruminants The content and composition of lipids in the plasma of new-born and adult ruminants are described in a previous article. 39a At birth, the plasma lipid content was found to be very low and the fatty acid compositions of individual lipid components resembled those of non-ruminant animals in a state of essential fatty acid deficiency. When newborn lambs were fed a normal milk diet for only 8 days, however, the plasma lipid compositions tended to resemble those of more mature animals. 18o The metabolic implications of the changes in lipid composition during this period are discussed at length by Noble.173b The plasma lipid compositions listed in Table 1 were obtained from calves on what might be considered a "normal" dietary regime, i.e. for the first 3 months of life they were milk-fed but wfth access to mixed pasture from weeks 2 to 3; at the end of this period, they were weaned onto a diet containing concentrates and lucerne. 229 A fully functional rumen would have developed during weeks 8 to 12. During the first 3 months when the calves were receiving milk containing 50~o or more of the energy as fat, plasma total lipid concentrations tended to rise. Cholesteryl esters, unesterified cholesterol and phospholipids all rose in concentration but triglyceride levels were variable and unesterified fatty acids tended to decrease in concentration. After weaning onto the comparatively low fat diet, there was a drop in plasma lipid concentrations. TABLE 1. Plasma Lipids~Changes in the Amounts (mg per 100 ml) of Total Lipid and of Individual Lipid Components in the Plasma of Calves with Age 229

Age

Total lipid

Triglycerides

Cholesteryl esters

Cholesterol

Free fatty acids

Phospholipids

Birth 2 days 6 days 10 days 30 days 3 months 4 months 6 months

140.0 250.7 295.0 348.3 392.0 401.3 312.0 314.2

6.8 33.5 25.0 29.3 13.4 22.6 23.5 21.5

37.2 67.5 103.3 131.8 161.3 159.0 128.7 118.3

8.0 13.3 18.2 19.9 24.0 23.3 18.2 15.5

20.7 9.6 8.1 9.1 7.9 6.3 4.3 4.8

50.9 110.7 129.2 149.4 168.8 179.2 159.7 142.4

248

W . W . Christie

A similar type of study has been performed with lambs. ~57 These changes would, ot course, be influenced by the time of weaning and by the fat or energy content of the diet, while the development of the rumen could be delayed by withholding access to roughage. When the animals are in the preruminating stage, the fatty acid compositions of plasma lipid components are particularly susceptible to dietary modification. During later development (9-16 months), i.e. the fattening period for beef cattle, the plasma lipid content, and in particular that of the neutral lipid components, tended to increase 34 although this was subject to variation with the energy content of the diet. t 54

B. Plasma Lipid Composition during Pregnancy and Lactation The compositions of the plasma lipids in non-lactating mature ruminants were discussed in a previous article. 39" The developing foetus and the functioning mammary gland have separate and distinct requirements for lipid that must be supplied by the intermediacy of the plasma lipids and these requirements are reflected in changes in the composition of the plasma lipids throughout pregnancy and lactation. Changes in hormone levels during these states and especially at parturition may be important in bringing about the lipid changes. There appear to be marked variations between animals in the effects of pregnancy and lactation on plasma lipid concentration and the results illustrated in Fig. 1 were obtained from a single cow 64 although most of the features that have been described by others 112"2°9 213 can be seen (whether all the observed changes were entirely typical is, however, uncertain). The lipid content of plasma tended to decrease 64 as pregnanc} progressed but there appeared to be an abrupt rise at, or immediately before, parturition after which there was a sharp drop with a subsequent increase as lactation progressed. During a similar time period, there was a steady decrease in the lipid content of plasma from non-pregnant cows. In late lactation, plasma lipid levels tended to drop again to values similar to those in non-lactating animals. A similar pattern was observed in the plasma of the ewe under the same conditions although the time-scale was somewhat different, i s 1 Although there appeared to be changes in the composition of the plasma lipids during pregnancy and lactation also, the literature on the subject is confused and there arc a number of conflicting reports possibly as a result once more of large variations betweer, animals. In one of the more systematic studies by Remond et al.. 2 ~2 the relative propor-tions of cholesteryl esters, triglycerides and phospholipids in the plasma of co~v,~; appeared to remain approximately constant throughout pregnancy (although the absolute amounts varied) but the free fatty acid concentration tended to rise as pregnanc.~ progressed. After parturition, there was a rapid rise in the free fatty acid content ol

E550 '~ 500

~ 450 400"

:35O'

-so -io -;o -2"o -,o Days

; .;o So So .;o .5"0 before or after

parturition

F1G. I. Variation in lipid concentration (mg per 100ml of plasma) with days before or after parturition in the plasma of a cow (redrawn from ref. 64).

Effects of diet on lipid composition

249

the plasma followed by a steady decline after the second week of lactation 64'212 but the relative proportions of cholesteryl esters and phospholipids remained fairly constant throughout the first 6 weeks of lactation while that of the triglyceride fraction tended to diminish. 212 Others have reported either no change 112,209 or a variable response 64'285 in the compositions of the lipids and lipoproteins of plasma during pregnancy and lactation in the cow. In plasma from pregnant ewes, lsl the free fatty acid content rose during the 3 days immediately before parturition although there was little change in the relative proportions of the other main components. At parturition, there were marked rises in the content of unesterified fatty acids and of phospholipids in plasma but these later tended to diminish as lactation continued. Similar changes in the concentrations of unesterified fatty acids in sheep plasma during pregnancy and lactation were found in a separate study. 142 The best documented of the changes that occurred during pregnancy and lactation were the small rise in plasma free fatty acid concentration just before parturition and the abrupt rise after parturition. 54'64'112'142"181'211'212'285 The former effect may be due to an increased requirement for substrate for energy production during the last stage of pregnancy while the latter effect is related to the heavy demand for fatty acids for milk fat synthesis at the onset of lactation. In each instance, free fatty acids are mobilized from adipose tissue in response to hormones; detailed studies of the phenomenon in ruminants appear to be lacking but it has been studied in rats. 23a As there is strong evidence that plasma triglycerides are the most important contributors of fatty acids for milk fat synthesis (see Moore and Christie166a), it might be expected that triglyceride concentrations in plasma would be closely correlated with the rate of milk fat synthesis and with stage of lactation, but this has not proved easy to establish experimentally. However, a positive curvilinear relationship between the concentration of plasma unesterified fatty acids and the yield of total milk fatty acids has been observed in lactating cows.~ 70 Little change was observed in the fatty acid compositions of individual plasma lipids during pregnancy but after parturition, the ratio of stearic to oleic acids in the triglyceride and free fatty acid fractions tended to increase as lactation progressed, 64'181'212'29° a change that was probably related to the amount of depot fat (comparatively rich in oleic acid) being mobilized. A similar effect was observed in the unesterified fatty acid fraction but there was little change in the composition of the phospholipids. In one study, the ratio of 18:2 (n-6) to 18:3 (n-3) fatty acids in the cholesteryl ester fraction of plasma apparently decreased markedly during pregnancy and lactation 64 but, in another study, 212 the opposite effect was found. Numerous analyses of plasma lipoproteins from lactating cows are available 33"a2'103'199'209'210"239-241'290'291 and can be compared with those of non-lactating animals described in a previous article. 39a

C. Dietary Fatty Acids and Plasma Lipid Composition For many years, one of the principal anomalies with respect to lipid metabolism in ruminant animals was that, despite the extensive biohydrogenation that occurred in the rumen, the lipids circulating in the plasma had a high total content of polyunsaturated fatty acids. Later it was shown that most of these components were in the cholesteryl ester and phospholipid fractions as opposed to in the triglycerides or unesterified fatty acids (see Christiea9a): As the latter two fractions were the most active metabolically, supplying fatty acids to many other organs such as adipose tissue or the mammary gland (see Moore and Christie 166~ and Vernon2aa~), this appeared to account for the comparatively low proportions of polyunsaturated fatty acids in these tissues. When diets rich in polyunsaturated fatty acids are fed in the normal way to ruminants, those unsaturated components that escape biohydrogenation in the rumen appear to be selectively taken up and esterified to the plasma phospholipid and cholesteryl ester fractions. Moore et al., 167 for example, found that when sheep were fed dried grass

250

W. W. Christie TABLE 2. Plasma Lipids Fatty Acid Compositions (Weight Percent of the Total) of Individual Lipid Classes of Sheep Given Diets of Hay or Dried Grass ~67 Cholesterol esters Fatty acid 16:0 16:1

18:0 18:1 18:2 18:3 20:4

Hay

Dried grass

14.3 2.8 3.5 35.3 33.3 5.1 2.8

13.6 2.8 4.1 33.8 28.1:' 10.2 ~' 2.S

Frec fatty acids

Triglyceridcs

Hay 32.8 4. 1

26.9 28.5 5.3 tr

Dried grass 28.4 ~' 4.3 37.3 b 24.5 3.2 Ir

Phospholipids

Ha}'

Dried grass

25.6 3.5 33.2 32.4 2.7

26.2 3.7 33.9 31.0 2.6

Hay

Dried grass

20.2 1.2 25.5 20.3 15.8 2.5 ;'.5

17.9 "* 1.3 2S,3" 20.4 i~2 ~' 54 h 71

•':hSigniticantly different (P < 0.01: P < 0,001) from the values obtained from the sheep on the hay diet.

(the fatty acids of which contained 57"_Jo linolenic acid), the plasma cholesteryl esters and phospholipids contained significantly more linolenic acid than was found in the corresponding lipids of sheep fed a hay diet (in which only 18010 of the fatty acids was linolenic acid) as shown in Table 2. On the other hand, there were no significant differences in the content of unsaturated fatty acids in the unesterified fatty acids or triglyceride fractions of plasma from sheep fed either diet although perhaps surprisingly, there was significantly more stearic acid in the plasma triglycerides from sheep on the diet of dried grass. Results analogous to these were obtained when various hay-grain mixtures were fed to lactating cows. 2°a Effects similar in nature although different in magnitude were also observed when fatty acid mixtures enriched in either linoleic or linolenic acids were infused for up to 5 days directly into the rumen of the sheep. I~'~ At the end of the infusion period, the polyunsaturated components that escaped biohydrogenation were again concentrated in the cholesteryl ester and phospholipid fractions of plasma. Much greater changes in composition would have been anticipated, of course. had similar experiments been performed with non-ruminants. Some results are shown in Table 3. On the other hand, when large amounts of linoleic acid were infused over a period of I hr directly into the abomasum of a sheep thereby avoiding the microorganisms of the rumen and any possibility of biohydrogenation, very different patterns of incorporation into lipid classes were observed t6u as illustrated in Fig. 2. Within 2 h r of the start of the infusion, there was a dramatic rise in the proportion of linoleic acid in the total fatty acids of the triglyceride and free fatty acid fractions of the plasma. These rises were reversed temporarily after 11-13 hr at a time when the sheep was given half its daily ration of hay. but then commenced to rise once more reaching a maximum m the triglyceride fraction after 30hr when the linoleic acid concentration began tL> drop. (A further minimum was observed in the proportion of linoleic acid in the frec fatty acid fraction, again corresponding to a feeding period.) In contrast, there ~as no observable change in the composition of the plasma phospholipids during the tirst 10 hr after the infusion was commenced but subsequently, there was a slow but stcadv TABU 3. Plasma Lipids Linoleic Acid Content (Weight Percent of the Total) of Individual Plasma Lipids of Sheep Before and After Daily infusion of Linoleic Acid (18:2) into the Rumen for 5 Days 16v 18:2 content

Lipid class Cholesteryl esters Triglycerides Unesterified fatty acids Phospholipids

Before infusion of 18:2

After infusion of 18:2

27.5 4.8 4.9 i 5.8

42.7 4.4 5.t) 26.1

Effects of diet on lipid composition ..' 40

.""

O~o 18:2

251

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~

.

j

,

~

.e-"

in each

~" "

lipid class

30 w

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e....~. • , I' • "

: '• •. . . | . ~ . . ~ • ' u " I

: . . . . 20 'm~m"~ " •

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lo

i

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,'o

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Time

(hours)

s'o

FIG. 2. Variation in the linoleic acid content (wt ~o of the total fatty acids) in each lipid class in the plasma of a sheep with respect to time after infusion of free linoleic acid into the abomasum. 168 T G = triglycerides, FFA = free fatty acids, P L = phospholipids, CE = cholesteryl esters.

increase in the linoleate concentration of this fraction until the end of the experimental period• A similar effect was seen with the cholesteryl ester fraction except that the rise in linoleate concentration was not observed until 24 hr after the start of the infusion. The physical form of the infused fatty acids did not appear to be important as analogous effects were observed when linseed oil or maize oil were infused into the abomasum instead of the free acids. In all the lipid classes, as the relative proportion of linoleic acid increased, there were compensatory decreases in the concentrations of palmitic and stearic acids. Although the results from the two sets of experiments may appear at first glance to be somewhat at variance, there were important differences in the manner in which the studies were carried out which may suggest an explanation of the phenomenon. Both were comparatively short-term experiments but in the second, in addition to the fact that the rumen was by-passed, much larger amounts of lipid were fed in a single dose and very much more polyunsaturated fatty acid must have reached the intestines over a short time period. This factor may be of critical importance as it suggests that, if excess polyunsaturated fatty acid is present in the intestines, the normal absorptive mechanism for these components is swamped (see also Noble 173a). Certainly, when large amounts of maize oil or free linoleic acid were infused into the rumen of a sheep in a single dose (40 g over 1 hr as opposed to 30 g over 2 hr) in further experiments described by the same authors, 179 the biohydrogenating effects of the rumen microorganisms appeared to be partly overcome and the pattern of incorporation of the linoleic acid into the plasma lipid classes with respect to time was similar to that obtained with the abomasal infusion. There was, however, a further delay of 1-2hr before the appearance of the excess linoleate in the plasma, presumably because of the additional time taken for fatty acids to pass from the rumen into the abomasum. Although the detailed mechanism of the phenomenon has still to be determined, a useful working hypothesis might be that, when sheep receive a normal low-fat foragebased diet, those polyunsaturated fatty acids that escape biohydrogenation are utilized preferentially by specific acyl transferases in the intestine for the synthesis of phospholipids in the intestinal wall membranes• Such phospholipids would turn over comparatively slowly, accounting for the 12 hr delay seen in the appearance of excess linoleic acid in the phospholipid fraction of plasma. This might also explain the delay in finding linoleate in the plasma cholesteryl ester fraction as the phospholipids may be intermediates in their synthesis via some enzyme system such as the lecithin-cholesteryl acyl transferase known to exist in plasma (see Noble]73~). On the other hand, the bulk of the saturated and monoenoic fatty acids reaching the intestines would be utilized rapidly for the synthesis of triglycerides which are absorbed and turned over quickly

252

w.w. Christie

in the animal. When large amounts of polyunsaturated fatty acids reach the intestines, the mechanism for segregating these components is not able to cope and the excess is utilized for triglyceride biosynthesis. Results that can be explained on the basis of the above hypothesis have also been obtained in numerous experiments in which polyunsaturated fatty acids, protected against the effects of biohydrogenation in the rumen by formaldehyde treatment, were fed to ruminants. In each experiment, the polyunsaturated fatty acid content of every lipid class in the plasma was augmented but such increases were most apparent in the triglyceride fractions. 43'46'62'182'226 On the other hand, when large amounts of untreated polyunsaturated fatty acids were fed to ruminants as a supplement to their normal diet (as opposed to being infused into the rumen in a single large dose), some may have escaped biohydrogenation in the rumen but little if any increase in the polyunsaturated fatty acid concentration of the plasma triglyceride fraction was observed.,~o.55.62,150.155.182,213,226.249 Although the most abundant single fatty acid circulating in the plasma of lactating cows was linoleic acid (up to 55'!,; of the total). less than 1% of this was in the triglyceride fraction, i.e. that fraction available for milk fat synthesis. The specific transfer of this acid to the plasma phospholipids and cholcsteryl esters may be a mechanism for conserving it for essential functions elsewhere in the animal. As formaldehyde-protected triglycerides would be hydrolyzed by pancreatic lipase rather than by the microbial lipases of the rumen, it might be expected that the resulting monoglycerides would be utilized for triglyceride biosynthesis, by the monoglyceride pathway in the intestine. However, no support for this hypothesis was obtained after structural analyses of lymphatic triglycerides of sheep fed protected fat supplements, ~'1 The effects of dietary supplementation with polyunsaturated fatty acids on the absolute amounts of individual lipid classes in the plasma of ruminant animals are less well documented and the published results are occasionally contradictory. The weight of evidence tends to indicate that all plasma lipids, with the possible exception of triglycerides which turn over rapidly, are elevated in concentration by feeding dietary supplements of unprotected polyunsaturated fatty acids to ruminants over comparatively short feeding periods (2--3 weeks) although possibly not over longer feeding periods. 42"t22"15°'155"191"249"286 Similarly, all lipid classes in plasma may be elevated in concentration by feeding protected polyunsaturated fatty acids. In particular, free cholesterol concentrations were raised two- or three-fold in lactating cows ~5''7,3°'~ and steers 92 although the effect was less pronounced in c a l v e s 292'293"299 and lambs. 5~'~°2 This appeared to be a consequence of increased cholesterol synthesis in the intestine and. in lactating animals, of decreased removal of cholesterol from the plasma by the mammary gland. 224 The effects of feeding dietary supplements of protected polyunsaturated fatty acids on the compositions of individual lipoprotein fractions of plasma from sheep and cattle have also been studied in some detail. °2'Is-' Considerable increases in the proportion of very low density lipoproteins and in the amount of triglyceride in this fraction were observed in comparison to values in control animals or in animals receiving supplements of unprotected polyunsaturated fatty acids. The changes in fatty acid compositions of individual lipid classes within the various lipoprotein fractions paralleled those found by others for whole plasma. It does not appear to be known whether the C20 and C22 polyunsaturated fatty acids such a s a r e found in cod-liver oil are subjected to biohydrogenation to any significant extent in the rumen although appreciable amounts of trans-isomers have been detected. 2°3 Certainly when dietary supplements of this oil were fed to lactating cows. the cholesteryl ester and phospholipid fractions of the 7-1ipoproteins of the plasma were elevated in concentration and contained appreciable amounts of longer-chain polyunsaturated fatty acids. 32'2°3 The effect was even more marked when the oil was infused into the abomasum. 2°3 On the other hand, the amount and composition of triglyceride fractions of plasma remained comparatively unaffected by such treatments.

Effects of diet on lipid composition

253

Polyunsaturated fatty acids did appear in the free fatty acid fraction of plasma and they must have been the principal source of those components that were ultimately found in the milk fat (see Section IV.B.4 below). 32 Although the major effort in feeding fat supplements to ruminant animals has been concerned in recent years with raising the levels of polyunsaturated fatty acids in products destined ultimately for human consumption, there have also been some studies of the effects of feeding more saturated fats to ruminants. For example, Moore e t al. 17° gave dietary fat supplements enriched in either stearic acid or palmitic acid to lactating cows and investigated the changes in plasma lipid composition that occurred. In each instance, the total fatty acid content of the plasma was raised although the relative proportions of the various lipid classes remained constant (free cholesterol was not determined, however). The stearic acid treatment gave elevated absolute levels of almost all fatty acids in plasma although the relative proportions of individual components changed somewhat and this was more evident in some lipid classes than in others. For example, in the plasma triglyceride and unesterified fatty acid fractions, the relative proportion of stearic acid was almost doubled while the concentrations of 16:0, 18:1 and 18:2 fatty acids were diminished in comparison to control values. In the plasma phospholipids, the concentrations of both 18:0 and 18:1 fatty acids were elevated while those of palmitic acid and most of the polyunsaturated components were diminished but in the cholesteryl ester fraction, the effects were rather small and could not have been readily anticipated, viz. a small decrease in the relative proportion of palmitic acid and compensatory increases in the concentrations of 18:3 and 20:4 fatty acids. Similar resultswere obtained in an analogous experiment with lactating goats. 55 When a dietary supplement of palmitic acid was fed to lactating cows, the absolute amounts of all fatty acids in plasma were again raised but the relative proportions of palmitic acid were increased and those of the Cls and polyunsaturated components tended generally to diminish in the plasma triglyceride, free fatty acid and phospholipid fractions in comparison with control values. The cholesteryl ester fraction was once more unusual in that the concentration of palmitoleic acid was greatly elevated while the relative proportions of 18:2 and 18:3 fatty acids were lowered. In a similar type of experiment, dietary supplements of lauric acid to lactating cows 214 produced elevated levels of cholesterol in plasma although the triglyceride and free fatty acid concentrations were not altered relative to control values but infusion of elaidic acid into the abomasum of lactating cows had no effect on plasma lipid composition. 215 Feeding of dietary supplements of longer-chain saturated fatty acids altered the relative proportions of the individual phospholipids in plasma increasing the concentration of phosphatidylcholine from 70~o of the total phospholipids with control animals to 88~o in those of the supplemented groups. 178 Compensatory decreases in the proportions of all the other phospholipids with the exception of phosphatidylserine were found. The changes in fatty acid composition of individual components were comparatively minor and with those animals receiving the stearic acid supplement, for example, there was a small increase in the concentration of 18:1 fatty acids and a similar decrease in the concentration of linoleate but no other significant changes in the phosphatidylcholine fraction. In the same fraction from animals receiving the palmitic acid supplement, the concentrations of 16:0 and 18:1 fatty acids were elevated while those of 18:0 and 18:2 fatty acids were diminished relative to the values found with low-fat control animals. The effects of supplementing the diets of fattening steers with various levels of comparatively saturated "animal fat" on plasma lipid compositions have also been studied in some detail. 61'154,155 The changes in fatty acid composition of individual lipid classes brought about by such dietary regimes appeared to be intermediate between those obtained by Moore e t al. 17° when palmitic and stearic acids were fed separately. The proportions of individual serum lipoprotein fractions were not affected by such diets. 6a Similarly, feeding of dietary supplements of protected tallow to sheep had very little effect on the composition of serum lipoproteins although the fatty acid compositions

254

w.w. Christie

of the triglycerides of the low and very low density lipoproteins were altered in that the relative proportions of 18:0 and 18:1 fatty acids were increased. ~s2 Though many facets of the relationship between dietary fatty acids and plasma lipid composition remain to be fully explained, such effects have important consequences for lipid metabolism in other organs as discussed later.

D. Heat and Cold Exposure and Plasma Lipid Composition There is a considerable amount of evidence to suggest that lipid metabolism in ruminant animals is affected by exposure of the animals to high environmental temperatures and one manifestation of this effect is an alteration in the composition of the plasma lipids. In particular, the concentrations of cholesterol, cholesteryl esters, free fatty acids and phospholipids were shown to be lower in the plasma of heat-stressed cattle than in that of control animals in a more normal environment. O'Kelly, ~85'186'~88'189 for example, demonstrated that the magnitude of the effect was dependent on breed and was more apparent in certain breeds of cattle from temperate climate zones (Bos taurus) at temperatures of 30-35°C than in Zebu cattle (Bos indicust which adapt more readily to such conditions. In consequence, seasonal variations in the plasma lipid compositions of cattle have been observed, tSv Further detailed studies of the phenomenon 1"~' demonstrated that exposure of cattle to a constant temperature of 35~'C produced a steady decline in the plasma total lipid concentration reaching a new equilibrium after 7 8 days when the concentrations of phosphatidylcholine, free cholesterol and cholesteryl esters were approximately 60°~, of the corresponding values in animals maintained at 22'C. In addition, the concentrations of linoleic acid in both the cholesteryl ester and phosphatidylcholine fractions were lower than those of control animals, a finding that may have been related to diminished lecithin-cholesterol acyl transferase activity in the plasma of heat-stressed animals. Similar changes in the concentrations of plasma lipid components were observed after, but not during, short-term exposure of cattle to heat stress when the animals had been returned to normal temperatures, tv5 As blood and plasma volumes were unaffected by such heat treatment, it appeared that the changes in plasma lipid composition were not caused by haemodilution but rather were a direct result of altered lipid metabolism, lvS'lv° Related effects on plasma lipid composition were found in cattle made hyperthermic by infusion of pyrogens into the bloodstream.19° Exposure to moderate cold has been shown to cause a marked elevation of the concentration of free fatty acids in the plasma of young lambs, 4 calves and steers; -'v8 the effect was less marked with adult sheep. 234 No other plasma lipid class was affected. 2"s The additional plasma free fatty acids were mobilized from adipose tissue to meet the increased energy demands under such conditions and the biochemical and physiological implications of the phenomenon were reviewed recently by Thompson. 277

E. Plasma Lipid Changes during Fasting When sheep 7'12~'211 and cattle 113 were subjected to fasting, there were rapid increases in the concentrations of the free fatty acids in the plasma of non-pregnant, pregnant and lactating animals. The rise was up to four-fold after only 24 hr and was up to ten-fold after 9 days. Most of this increase was probably the result of mobilization of fat by lipolysis of adipose tissue in order to supply the energy requirements of the animals. During fasting, the proportions of 16:0 and 18:0 fatty acids in adipose tissue lipids tended to decrease for the first 9 days while that of 18:1 tended to increase but these trends were later reversed, lzl The amounts of all the principal fatty acids in the free fatty acid fraction of plasma with the exception of 18:2 increased roughly in proportion. In the neutral lipid fraction of plasma, the proportion of 18:1 relative to 18:2 fatty acids increased.~21 Although there were also increased free fatty acid con-

Effects of diet on lipid composition

255

centrations in the plasma of lactating cows, ~13 no increase in the uptake of free fatty acids was observed. Fasting was also shown to lead to a considerable diminution in the rate of production of volatile fatty acids, particularly acetate, in the rumen and this was reflected subsequently by diminished acetate concentrations in the plasma 6'1°'23'113 and presumably by a reduced rate of fatty acid synthesis de novo in the tissues. III. DIET AND OTHER FACTORS AND THE LIPID COMPOSITION OF ADIPOSE TISSUE AND SKELETAL MUSCLE A. Lipid Composition in Adipose Tissue and Skeletal Muscle of Young and Developing Ruminants Adipose tissue of ruminant animals, as that of most other species, consists almost entirely of triglycerides with only small amounts of unesterified fatty acids and other lipids. Skeletal muscle is generally heavily infiltrated with adipocytes and with intracellular free lipid droplets that consist largely of triglycerides but, in addition, there are appreciable amounts of phospholipids that are constituents of the membraneous structure of muscle (see Christiea9a). Adipose tissue is a major site for fatty acid synthesis de novo and for desaturation of stearic to oleic acid in ruminant animals but a large proportion of the total fatty acids in the organ will be of dietary origin and is derived from the triglyceride and unesterified fatty acid fractions of plasma (the enzyme lipoprotein lipase is essential for this process). Dietary effects on the composition of these key plasma lipids were discussed above. The utilization of dietary fats by young ruminants was reviewed in 1971.28o The lipid composition of tissues of new-born ruminants is described in detail elsewhere39a,173b so need only be discussed briefly here. Perinephric and subcutaneous adipose tissue triglycerides in neonatal calves95 and lambs 94 and perirenal triglycerides in new-born lambs 17'~ were characterized by high levels of oleic acid (52-66Vo of the total) together with appreciable amounts of palmitiC and stearic acids. No linoleic acid or trans-unsaturated fatty acids and trace amounts only of odd-chain and branchedchain fatty acids were found. After 2 days on a normal diet of ewes' milk, small amounts of linoleic acid and trans-monoenoic acids appeared in the adipose tissue of lambs while after 8 days, the amount of oleic relative to stearic acid diminished appreciably. 174 By the age of 4-6 weeks, if the lambs had access to roughage, the rumen was developed and the fatty acid composition of the adipose tissue tended to resemble more and more that in adult animals. ~6,4s'z75 The same was true of calves. 72'75"95'233'299 When the rumen is fully functional, the fatty acid composition of adipose tissue does not normally respond rapidly to changes in the lipid composition of the diet because of the biohydrogenating effect of the rumen and of the selectivity of esterification of the unsaturated fatty acids that escape this process to the plasma components, namely cholesteryl esters and phospholipids (see Section II above) which are not hydrolyzed by lipoprotein lipase. Some comparatively minor changes in the lipid composition of adipose tissue and skeletal muscle of young ruminants were found to occur with age or in response to changes in the composition of the basal diet when the rumen was fully formed and these changes were influenced by season, by the sex of the animal or by other variables that were not easily controlled. 4a It has been reportedthat the ratio of 16:1 to 18:0 fatty acids in adipose tissue of cattle increased linearly with age. 76 If young ruminants are denied access to roughage, the rumen does not develop and the fatty acid compositions of the tissues change in response to the composition of the dietary lipids in a manner similar to that found in non-ruminants. For example, Siren 233 showed that, if calves were fed 6~ linseed oil in a milk replacer and were not given hay in the diet, the perinephric fat contained 10--11~o lirrolenic acid after 30 days. On the other hand, there was only 0.6--1.8~o of this component in the perinephric fat of calves fed a similar milk replacer but with access to hay ad libitum. Toullec

256

W.W. Christie

and Mathieu 279 demonstrated that pre-ruminant calves, given milk substitutes.containing tallow, lard, groundnut, coconut and palm oils until they were 95 days old, laid down adipose tissue and muscle fats that were influenced markedly by the compositions of the diet. However, shorter-chain fatty acids such as lauric acid were not deposited rapidly as such, but were chain-elongated, principally to myristic acid to a considerable degree first, although the site of this chain-elongation step does not appear to be known. Calves fed methyl myristate itself as 10"; of the dry matter of a substitute milk. laid down a depot fat that after 82 days contained 28.9!,.; myristic acid. a value approximately eight-fold higher than in a similar group of animals on a linoleatc-supplemented dim: the linoleic acid content of the adipose tissue was correspondingly elevated in the latter instance. 8° Dietary stearic acid was desaturated in large measure to oleic acid by the time it was deposited in adipose tissue. Findings similar to these havc been recorded by others l¢''~'3'~¢'5"2s°2s,288 and some results from a representative experiment aic listed in Table 4. TABLI 4. Adipose Tissue Lipids Fatty Acid Compositions (Weight Percent ot the Total) ot Dietary Fat Supplements in Milk Substitutes and in the Subcutaneous Adipose Tissue of 90-Day Old LambsI'' Fat supplement Diet Ewes" milk Coconut oil Tallow Palm oil Adipose tissue Ewes' milk Coconut oil Tallow Palm oil

12:0

14:0

Fatt) acid composition 16:0 16:1 18:0

18:l

t8:2

6.6 35.3 1.4 0.5

13.2 13.5 3.6 1.6

32.0 16.9 28.2 45.9

0.O 3.0

10.7 4.4 23.,4 5~

I7,~ 14,1 36.2 39.5

1.0 4.6 3.2 7.{)

1.0 4.6 0.6 0.3

7.7 13.3 2.6 1.4

29.8 29.1 25.4 26.8

4.5 4.g 5.3 5.6

10.I 5.3 I1.6 ,<3

3S.¢* 37,0 50,9 467

1.2 2.7 2.4 3.5

On the other hand, when young ruminants were fed such widely differing oil supplements but at the same time had access to roughage, such treatment differences in the fatty acid composition of the adipose tissue as were found tended to be comparatively minor. 1<48"2~5 Less c o m m o n fatty acids, such as the C2o and ('22 components of partially hydrogenated fish oils, were incorporated rapidly into the depot fats of calves when included in the diet. 89 A high proportion of the work on fat supplementation of the diets of young ruminants has been concerned with elevating the polyunsaturated fatty acid content of tissues destined ultimately for human consumption. For example, Wright c t a / . 3°1"3°2 demonstrated that lambs could retain the suckling reflex for up to 16 weeks and that calves and lambs fed milk replacers containing 12.5-70 g sunflower oil per day without roughage in this manner developed muscle and adipose tissue fats that contained up to 40,}0 linoleic acid in the triglyceride fraction after 30 days. At the higher levels of sunflower oil supplementation, the rate of growth of the lambs relative to that of control animals was depressed. 3°2 Others 72.75.299 have fed linoleate-supplemented milks to bull calves for shorter periods (10 weeks), then transferred the animals to solid diets containing linoleate protected against ruminal biohydrogenation by casein-formaldehyde treatment. At 17 weeks of age, adipose tissue (tailhead) biopsies from calves fed such a diet contained up to 12.8°/£ linoleic acid, whereas adipose tissue from calves fed an unprotected casein-linoleate diet for the same period contained only 3.6% linoleic acid.

B. Dietary Fatty Acids and Adipose Tissue and Skeletal Muscle Composition in Adult Ruminants The rumen has a major effect on the composition of the tissue lipids in that it is the site of hydrogenation of a high proportion of the dietary unsaturated fatty acids but it is by no means certain that this is the only circumstance in which it exerts an

Effects of diet on lipid composition

257

influence. There has been little work on the incorporation of shorter-chain dietary fatty acids (such as lauric acid) into adipose tissue of mature ruminants but there is no reason to suspect that it would be otherwise than with young animals as described above. Dietary supplements of octanoic and decanoic acids had very little effect on the composition of adipose tissue lipids at three sites in sheep relative to that of control animals. 56 The myristic acid content of adipose tissue of sheep was raised four-fold (from 4.2 to 15.5~o) when 60 g of the methyl ester derivative of this compound was infused daily into the rumen of sheep over a 28-day period and there was a concomitant decrease in the proportion of oleic acid in the tissue. 8t On the other hand, when similar amounts of methyl myristate were infused into the abomasum, there was virtually no effect on the composition of the adipose tissue lipids. In the latter instance, the digestibility coefficient of the methyl myristate was much lower than in the former (68.9 as opposed to 94~o) but the reasons for this are unknown although an absorption effect of the kind described by Harfoot 11lb may have played a part, since a high proportion of the fatty acids of the rumen bacteria was myristic acid (44.8~ versus 14.1~o in the control animals) when this compound infused into the rumen. The fate of that myristic acid absorbed from the intestines when it was infused into the abomasum was also obscure; conceivably, it was subjected to oxidation or was esterified to plasma components such as cholesteryl esters and phospholipids that did not supply fatty acids to adipose tissue. Further work is obviously needed to clarify the point. There do not appear to be any systematic studies of the effect of supplementation of ruminant diets with saturated fatty acids of longer chain length than C14 on the composition of adipose tissue lipids, although related experiments, in which the compositions of milk lipids were examined, have been performed with results that may have relevance to adipose tissue and these are discussed in Section IV.B. However, there have been a number of studies in which animal fats or tallow, that contained comparatively high proportions of saturated and monoenoic fatty acids, were fed as dietary supplements to ruminant animals. In one of the first systematic feeding experiments in which modern chromatographic methods were used for analysis of the tissues, TM steers were fed a basal diet or one supplemented with either 2.5 or 5~o animal fat. The supplemented diets contained appreciably more palmitic and stearic acids than did the basal diet but, after 22 weeks, the only significant (P < 0.05) effect on the composition of the rib fat was a small increase in the content of stearic acid with increasing levels in the diet. Some of the results are listed in Table 5. Similar results were obtained by others. 4°'28~ SurPrisingly, when high levels of trans-fatty acids in partially hydrogenated vegetable oils were fed in dietary supplements to sheep, considerable amounts appeared to escape biohydrogenation and were incorporated into the adipose tissue lipids. T M Roberts and McKirdy 2~6 fed steers a basal diet or one supplemented with either rapeseed oil, sunflower seed oil or animal tallow for 19 weeks but found no significant differences in the relative proportions of the various C~8 fatty acids in the perirenal fat from any of the groups (see Table 6). This was presumably due to the extensive biohydrogenation of the fat supplements in the rumen prior to absorption as is discussed further below. The fat from animals receiving the rapeseed oil supplement contained TABLE 5. Adipose Tissue Lipids--Fatty Acid Compositions IWeight Percent of the Totali of Dietary Fat Supplements and of Rib Fats of Steers After 22 Weeks TM

Fat supplement (~/o animal fat) Diet 0 2.5 5 Rib fat 0 2.5 5

Fatty acid composition 16:1 18:0 18:1

14:0

16:0

18:2

0.2 1.6 2.5

17.7 30.3 35.2

1.0 2.8 2.7

4.3 12.0 14.1

38.8 42.6 42.5

36.4 10.7 3.1

3.5 3.7 3.3

31.8 30.6 30.6

4.6 5.0 3.4

14.5 16.8 21.4

42.2 41.1 40.0

1.2 0.7 0.3

258

W . W . Christie

TABLE 6. Adipose Tissue L i p i d s - - F a n y Acid Composition (Weight Percent of the Total) of Diets and the Resulting Perirenal Fat of Steers Fed a Control Diet or O n e Supplemented with Either Rapeseed Oil, Sunflower Seed Oil or Animal Tallow z ~ Fatty acid composition Ration Diet Control Rapeseed ~ Sunflower seed Animal tallow Perirenal fat Control Rapeseed" Sunflower seed Animal tallow

14:0

16:0

16:1

18:0

18:1

18:2

1.1 0.4

2.3

21.7 9.0 11.0 27.2

3.5

3.1 1.9 3.3 15.3

31.6 30.0 25.0 33.4

42.5 28.8 60.7 144

3.2 3.0 2.8 3.2

24.8 21.1 20.8 25.8

2.3 2.1 2.0 2.9

29.4 28.8 31.9 29.9

35.5 34.3 37.1 32.6

L0 1.7 2.8 2.6

20:1

22:1

8.1

14.4

~5

~2

~'7.4!~,~, linolenic acid was present in the diet but only trace a m o u n t s were found in the perirenal fat

some eicosenoic and erucic acids; it is not known whether these components were hydrogenated in the rumen and then desaturated in the tissues before esterification or if they simply passed through the rumen unchanged. More recently, 6°'63 the effects of feeding a conventional basal diet or one supplemented with either 6°~o animal fat or safflower, oil on the composition of adipose tissue and skeletal muscle fats from a number of sites in steers were studied and the changes observed were correlated with changes in plasma lipid composition.l ~,155 In fact, such changes as were observed were very minor; the proportions of palmitic and stearic acids in all tissues of animals receiving animal fat supplement were slightly higher than in animals receiving the other two rations but the differences were rarely statistically significant (P < 0.5). Related changes in the compositions of the plasma triglyceride and free fatty acid fractions were discussed earlier (Section II.Ct. In a second experiment, animal fat supplements were fed at three levels (5, 10 and 15°o) in addition to the basal diet but statistically significant increases in the relative proportions of saturated to monoenoic fatty acids with increasing levels of dietary fat were found in only two of the ten tissues studied. There was no change in the amount of fat deposited with dietary treatment in either of the experiments. Typical ruminant diets consist of forage crops such as grass, alfalfa or clover (either as pasture or in some conserved form) and of concentrates, i.e. high energy cereals such as maize or barley. Neither tends to contain a high proportion of lipid and that present consists mainly of esterified polyunsaturated fatty acids, linolenic acid being the main component of forages and linoleic acid of cereals. Also, forages contain a much higher proportion of fiber or roughage than do the concentrates. T o r e and Matrone T M observed that sheep fed a purified low-fiber diet produced low melting depot fats that contained lower proportions of palmitic and stearic acids and more palmitoleic and oleic acids than did sheep on a more normal diet. Similar findings have been recorded by others, t6°'27°'3°6 However. it is unlikely that these changes were the result of variation in the fatty acid composition of the diet but rather were a consequence of altered tureen fermentation (see Section IV.C). The lipids of forages or of concentrate supplements in ruminant diets may have some influence on adipose tissue composition in the animals as minor but statistically significant differences were found on varying the nature of these components in the diets. 36'4°'4~ When these basal diets are supplemented by polyunsaturated fatty acids in vegetable oils, there is little effect on the fatty acid composition of the adipose tissue as the small proportion of the unsaturated components that escapes biohydrogenation is not found in the plasma triglyceride and free fatty acid fractions, i.e. those fractions that are most readily available to the peripheral tissues as has already been described above. The results listed in Table 6 illustrate the point and other papers to this effect have been cited in this and the previous section. Numerous reports of a similar kind exist

Effects of diet on lipid composition

259

in the literature. 37"42'58'115.130.156,161,282 Exceptions are occasionally reported, however. For example, vegetable oils fed with diets containing high amounts of barley have been found to give depot fats with elevated levels of linoleic acid. .1"98 It seems possible that, in these circumstances, different populations of bacteria and protozoa arise within the rumen with a diminished capacity for biohydrogenation. As stated earlier, when polyunsaturated fatty acids are infused into the abomasum or intestines of ruminant animals, they are not subject to biohydrogenation and may be absorbed in such amounts that they cannot be esterified solely to the plasma cholesteryl ester and phospholipid fractions but appear in appreciable amounts in the triglyceride and free fatty acid fractions also. They thus become subject to lipoprotein lipase activity and can be taken up by the adipose tissue. The linoleic acid content of adipose tissue lipids of sheep and cattle has been greatly elevated in this way. 58'81'156'184 Although infusion techniques of this kind are exceedingly useful for experimental purposes, they are not suitable for mass production of ruminant animals with depot fats rich in polyunsaturated fatty acids under commercial farming conditions. The most useful procedure in the latter instances consists in the use of vegetable oils protected against digestion in the rumen by formaldehyde-treated casein or oil-seeds (as described earlier in Section I) as supplements to the basal diets. For example, Scott eta/. 226 obtained ten-fold elevations of the linoleic acid contents of the perirenal and subcutaneous adipose tissue of sheep (Table 7) by feeding supplements of safflower oil protected by formaldehyde-treated casein. There were compensating decreases in the relative proportions of most of the remaining fatty acids. Results of a similar nature have been obtained by numerous other workers by means of analogous procedures in both adipose tissue and skeletal muscle of sheep 1'13"14'44'84"90'115"117'123"161 and of cattle.,~3, 57,83,91,92,118,130, 161,173,272.292 Some site variations in the relative proportions of linoleic acid deposited in the tissues have been observed but such differences tended to be minor and only in the intramuscular brisket fat was there appreciably less than in other tissues.118 TABLE 7. Adipose Tissue Lipids--Effects of Feeding Formaldehyde-treated Safflower Oil-Casein Supplements on the Fatty Acid Composition (Weight Percent of the Total) of Depot Fats from Sheep 226 Perirenal fat

Subcutaneous fat

Fatty acid

Unsupplemented basal diet

Basal plus protected supplement

Unsupplemented basal diet

Basal plus protected supplement

14:0 16:0 16:1 18:0 18:1 18:2 18:3

2.8 18.2 3.3 28.4 37.2 2.8 1.2

2.0 14.8 1.8 24.0 24.7 28.9 1.0

2.8 19.2 3.7 18.2 45.4 3.2 1.9

2.2 I 1.1 3.4 10.1 38.8 28.1 2.7

Stereospecific analyses showed that high proportions of the additional linoleic acid incorporated into the adipose tissue of ruminants by such means were preferentially esterified to position sn-2 and also, though to a lesser extent, to position sn-3 of the triglycerides. 115'~61 Over the range of samples examined, there appeared to be a linear relationship between the total linoleic acid content of the triglycerides and the proportion found in position sn-2.11s The oxidative stability 75 and the flavor properties 9°,91 of meat from animals receiving polyunsaturated oil supplements protected by formaldehyde treatment have been studied but detailed discussion is outside the scope of this review. The effect of such dietary supplements on lipogenesis in ruminant animals has been reviewed elsewhere. 22'*,288a Dietary fat supplements, in general, appeared to have variable effects on the rate of growth of ruminant animals where many other factors were. important. Again, such effects are not considered here.

260

W, W. Christie

C. Branched-chain Fatty Acids in Adipose Tissue

Iso- and anteiso-methyl-branched fatty acids are ubiquitous if minor components of the tissues of ruminant animals and have their origin in the rumen where they are synthesized de novo by bacteria (see Harfoot 11 lb). More recently, G a r t o n and co-workers have shown that, under some circumstances, appreciable amounts of a wide range of different branched-chain fatty acids, in addition to greater amounts of normal odd-chain fatty acids than usual, can accumulate in the adipose tissue of ruminants. In particular. lambs fed diets containing a high proportion (up to 90°~) of barley laid down subcutaneous depot fats containing high proportions of such compounds as is illustrated by the results shown in Table 8. 68 One consequence of this unusual composition was that the depot fats were particularly low melting. Similar results have been obtained with goats 7° but apparently not with cattle or with red deer. °3 High resolution ~as chromatography in conjunction with mass spectrometry was used to identify many of the compounds present showing them to be a complex mixture of at least 125 mono-, diand tri-methyl substituted components. 65'66'236 Monomethyl-branched fatty acids of chain-length from 10 to 17 carbon atoms constituted the greatest proportion of these and within each molecular species the methyl branch could occur on almost any of the even-numbered carbon atoms. TABLE 8. Adipose Tissue Lipids Fatty Acid Composition (Mol % of the Total) of Perinephric and Subcutaneous Adipose Tissue Triglycerides of L a m b s Fed Barley-rich Diets or Grass Cubes "8 Fatty acid composition

Diet and tissue Barley-rich Perinephric Subcutaneous Grass-cube Perinephric Subcutaneous

14:{)

16:0

16:1

18:{)

18:1

18:2

Odd-chain n-acids

Branchedchain acids

4 4

27 23

2 l

19 10

35 42

5 5

4 ~

2 9

2 2

19 21

2 3

36 14

30 46

3 4

3 4

2 2

It appears likely that these methyl-branched fatty acids were produced as a consequence of the substitution of methylmalonic acid for malonic acid during fatty acid synthesis de novo in the tissue (subcutaneous adipose tissue contains a higher proportion of fatty acids synthesized in the tissue than does the perinephric fat). Methylmalonic acid was in turn derived from propionic acid which is produced in greater amounts than is usual in the rumen of barley-fed animals. 196 Evidence to support this hypothesis has come from a considerable body of w o r k 6 7 " 6 9 ' 9 3 ' 1 4 6 A 9 5 ' 1 9 ° ' 2 2 1 which is discussed in detail elsewhere. 173b It may be relevant to note here that when propionic acid was infused into the rumen, of sheep, high proportions of branched-chain fatty acids were deposited in the adipose tissue but this was not so when acetic or butyric acids were u s e d . 96,97

The effect of high barley diets on tissue fatty acid compositions did, however, appear to be variable as high levels of branched-chain components were not always obtained in adipose tissue of sheep on such diets. 41"98'143"172 It seems probable that different populations of rumen bacteria and protozoa arise in some circumstances that produce less propionic acid than others. T h e occurrence of trace amounts of phytanic and related acids, products of phytol metabolism in the rumen, in the adipose tissue of ruminants was discussed in an earlier article s9a and has been reviewed by Lough. a44 Although it was shown that the triglyceride fraction of plasma from cows on a silage-based diet could contain up to 13~ phytanic acid, very little of this was incorporated into adipose tissue triglycerides; lipoprotein lipase was possibly unable to hydrolyze esters of such highly branched acids effectively. 64,14- 5

Effects of diet on lipid composition

261

IV. DIET AND OTHER FACTORS AND THE YIELD AND COMPOSITION OF MILK FAT

A. The Effect of Breed, Staoe of Lactation and Season on Milk Fat Composition The yield and fatty acid composition of milk can be affected by any number of factors, many of which are interactive and some of these have been reviewed elsew h e r e . 124'136'217"218'222"251 A s a consequence, it is not always easy to identify the cause of a given small change in milk fat composition although some of the more important factors have been characterized. For example, differences in the content and fatty acid compositions of milk fat from different breeds of cattle have been o b s e r v e d 124'135'217"268'294 and comparisons between single-egg and two-egg twins showed that these features were under genetic control. 73 The changes in the overall composition of milk associated with the lactationai cycle of the diary cow have been much studied and reviewed. 124'217'218'25t Certain of the changes that occur are mediated by husbandry practices that can confuse the interpretation of results. In numerous studies, for example, it was found that, while the absolute amount of fat secreted by the bovine mammary gland decreased throughout lactation, the relative proportion of fat in milk was maximum in early lactation then fell to a minimum value at about 10 weeks post-parturition before increasing once more (reviewed by Rook 2~7). This final increase in fat concentration may have been a result of a commercial practice of increasing the proportion of high energy concentrates in feed-stuffs during late lactation. On the other hand, when the energy consumption of the cow was altered simply to conform with its requirements for maintenance and production, the proportion of fat in milk increased slowly but steadily throughout lactation. 269 In contrast, the absolute amount of milk fat secreted decreased linearly with time throughout lactation from about 0.9 kg per day at the start to 0.6 kg per day at the 40th w e e k . 269 Similar results have been reported by others. 5a Some marked changes in the fatty acid composition of milk fat have also been observed during the lactational cycle and, while some of these may be related to dietary or climatic factors, others are apparently a consequence of changes in the rate of fatty acid synthesis in the mammary gland under the probable influence of hormone action. For example, Senft and Klobasa 227 studied the changes that occurred during the first 15 days of lactation and found that the colostral milk secreted during the first day of lactation contained a lower proportion of short-chain fatty acids, especially butyric acid, and more palmitic acid than was found on subsequent days. (Similar findings were obtained in detailed analyses of pre-milk from the mammary gland of cows near to parturition. 3°3) After the first week or so of lactation, there is some dispute as to the nature of the changes that OCCUr s3'54'199"200'227'268'269 but the general, if not unanimous, conclusions are that the relative proportions of short-chain fatty acids, with the possible exception of butyric, increased for the first 8-10 weeks of lactation, that the proportion of palmitic acid remained relatively constant but that the concentrations of stearic and octadecenoic acids tended to diminish during this period. After the tenth week of lactation, such compositional changes as were observed tended to be comparatively minor although there may have been a slight reversal of the earlier trends. In most studies, little change in the relative proportions of linoleic and linolenic acids that could be associated with the stage of lactation was found and when any significant changes were observed, they appeared to be due to dietary factors. The results for the first 10 weeks of lactation, particularly those of Decaen and Adda, 53 appeared to be a consequence of the relatively constant rate of synthesis of short-chain fatty acids during a period when total milk fat secretion was declining and the absolute amounts of only the longer-chain fatty acids that were secreted diminished. After the tenth week, there was a progressive decrease in the rate of synthesis and secretion of the shorter-chain fatty acids also, so that the relative proportions of all fatty acids J.P.L.R. 1 7 3

a

262

W . W . Christie TABLE 9. Milk Lipids--The Effect of Season on the Fatty Acid Composition (Weight Percent of the Total Fatty Acids) of Milk 2°2

Fatty acid 4:0 6:0 8:0 10:0 12:0 14:0 16:0 16:l 18:0 18:1 cis 18:1 trans 18:2 + 18:3

Winter (March)

Summer (June}

3.5 1.4 1.1 2,7 3.9 12.7 34.4 1.3 11.6 19.9 2.5 1.5

3.6 1.3 0.9 2.4 2.7 9.8 25.4 0.9 15.8 24.3 6.4 1.9

remained comparatively constant. During early lactation, there is extensive mobilization of fatty acids from adipose tissue and these acids would comprise mainly Cls components. The gradual fall in the rate of secretion of these fatty acids during lactation probably reflects a drop in the amount of lipid mobilized. There have also been a number of studies of the effect of season on the fatty acid composition of milk. 11,29.102,105.110.114,119,120.125.159,198,202.228 Again, a number of factors can have an effect on the results obtained and these include stage of lactation, general animal husbandry, diet and climate so that occasionally conclusions were reached that were apparently contradictory. However, the general weight of evidence tended to suggest that milk from cows in winter contained a higher proportion of palmitic relative to stearic and octadecenoic acids than milk from the same cows in summer. The results listed in Table 9202 were typical of many of those that were available. A change in the diet of the animals in spring from hay-concentrate mixtures to fresh pastures, that contained relatively high proportions of linolenic acid in the plant tissue lipids, was probably the single factor with the greatest effect on the phenomenon, as the proportion of linolenic relative to linoleic acid in milk also tended to increase in s u m m e r . 1 1 " 2 9 " 1 ° 2 " 1 ° 5 . 1 2 ° ' 1 2 5 In addition, the proportion of trans-monoenoic, conjugated dienoic and related fatty acids that arise during the incomplete biohydrogenation of dietary polyunsaturated fatty acids in the rumen (see Harfoot t11b) tended to increase during the summer months. 11,102,140.202 It is well known (reviewed by Rook 217) that the yield of milk fat from a cow decreases with the number of lactations but there is no effect on the fatty acid composition of milk fat. 228 Although the interval between milkings had an effect on the yield of milk fat 218 and on the relative proportions of triglycerides and phospholipids, there appeared to be no change in fatty acid composition. .27 There was no difference in the fatty acid composition of milk fat from cows milked in the evening as opposed to the morning. 199'228"269

B. Dietary Fatty Acids and the Yield and Composition of Milk Fat 1. Some General Considerations When the lactating cow is on a normal high roughage herbage/cereal-based diet, the daily output of milk fat is approximately the same as its daily intake of lipid. It has appeared to many investigators over the last 60 years that supplementation of the diets of lactating cows with various fats and oils might lead to improvements in milk fat yield or to modifications of the composition of the milk fat of potential benefit to the consumer. Much of the early work on the subject has been discussed in reviews

Effects of diet on lipid composition

263

by others; 169'251'271 this review covers only the more recent systematic studies made possible by the development of gas chromatography and its application to the determination of the fatty acid composition of lipids. In comparing various dietary supplements, the choice of a suitable control treatment is important. The lipid composition of the milk from cows on a normal forage/concentrate-based diet, which in many studies has been considered an appropriate control diet, is discussed in a previous article 39a but for some purposes, it may be necessary to have control treatments in which the diet is depleted in lipid. In this circumstance, a higher proportion of the milk fatty acids are produced by synthesis de novo within the tissue and this is reflected in the composition of the milk fat which tends then to contain only low amounts of the Cla fatty acids and up to 50% palmitic acid. 2°'150.289 Also, the yield and composition of milk fat can be profoundly influenced by the amount and nature of the roughage component of the basal diet (see Section IV.C) and this can lead to discrepancies in the results from apparently similar experiments. Differences in the nature and activity of the rumen microflora can cause similar difficulties. Dietary fats can alter the fatty acid composition of milk fat in a number of ways. For example, a given fatty acid may be absorbed and transported to the mammary gland where it is esterified unchanged and so appears in the milk fat in a higher proportion than is usual. Alternatively, it may be altered in the rumen by hydrogenation, for example, and eventually be esterified in the hydrogenated form in the mammary gland or it may be further modified by desaturation before esterification, i.e. the fatty acid deposited in milk fat may bear little apparent relationship to that in the diet. In addition, larger amounts than usual of particular fatty acids in the diet may affect lipid metabolism in the animal by various means. For example, the uptake of fatty acids by the mammary gland may be inhibited or there may be some inhibition of one of the enzymes involved in fatty acid synthesis. Dietary long-chain fatty acids can also affect volatile fatty acid metabolism in the rumen reducing the availability of the low-molecular weight substrates required for fatty acid synthesis. Some information on the effects of particular dietary fat supplements on milk fat synthesis or on general lipid metabolism can be obtained from analyses of the daily yields of individual fatty acids in milk fat. There may, for example, be increased uptake and secretion of the dietary fatty acids but diminished synthesis of fatty acids de novo so that there is little effect on the overall milk fat yield. It is generally assumed that the C4-C14 fatty acids are entirely of endogenous origin (i.e. synthesized in the mammary gland itself) and that the C~8 fatty acids are entirely of exogenous origin while the C16 components mayhave both endogenous and exogenous origins. Alterations in the amounts of specific components relative to those in milk fat from control animals can provide a clue to the site or mechanism of any inhibition. Any conclusions drawn solely from such analytical data, however, must be to some degree speculative. When information is also available on the composition of the plasma lipids or of the rumen volatile fatty acids in the same experiment, .firmer conclusions can be drawn. 2. Saturated and Monoenoic Fatty Acids

Although small amounts of acetic acid have been found in esterified form in milk fat (see Christie39~), all studies of the effect of supplementation of the diets of ruminants with acetic acid have been concerned with the rates of secretion of longer-chain fatty acids. For example, when acetic acid (up to 1500 g per day) was infused directly into the rumen of lactating cows, the yield of milk fat was increased219'22°'261 and, in particular, the amounts of the C4-C16 fatty acids secreted daily increased although the yield of CIa fatty acids decreased. 261 Similar but less pronounced effects were observed when smaller amounts of acetic acid (500-750g per day) were infused intravenously;262 in this instance, palmitic acid was the only component that increased appreciably in concentration. The increased rate of secretion of C4-C16 fatty acids was probably a result of increased availability of substrate for the mammary fatty acid synthetase. 261'262 As

264

W.W. Christie

the C18 fatty acids would be largely of dietary origin, it might be argued that the diminished secretion of the C~8 fatty acids was the result of some effect of increased acetate concentrations on the concentrations of the plasma triglyceride or free fatty acid fractions; no consistent effects were observed, however. Butyric acid infused intraruminally or intravenously into lactating cows produced similar effects to those of acetate on the total yield of milk fat and on the yields of individual milk fatty acids. 219'22°'26t'262 Presumably the increased availability of /~-hydroxybutyrate would be a factor in stimulating synthesis of fatty acids de no~'o in this instance. Tributyrin infused intravenously caused a drop in the rate of secretion of palmitic and oleic acids. 267 On the other hand, somewhat different effects were observed when dietary supplements of acetic acid (or its sodium salt) were fed to goats. For example, when 58 g per day of acetate was supplied in this way, the yields of all fatty acids, but expecially of the C18 components, were increased. 85 When the supplementation was increased to 100g per day, only the amounts of the C18 fatty acids secreted increased. 1°4 In these experiments, the concentration of the plasma free fatty acid fraction, that supplies a proportion of the C~s fatty acids for milk fat synthesis, tended to increase under acetate supplementation. No explanation is available for the observed differences in acetate metabolism between cows and goats. Supplements of propionic acid administered intraruminally or intravenously to cows tended to lower the yield of milk fat, the magnitude of the effect being dependent on the amount infused, by effecting a diminution in the amounts of all the fatty acids secreted with the exception of that of palmitic acid. 59"88"219'220'261'262 NO changes in the concentrations of individual plasma lipids were observed but the concentrations of plasma triglycerides recorded were low and variable. 26~'262 Although propionic acid is glucogenic in its metabolism, the response was somewhat different from that when glucose was infused intravenously. 88'262 Tripropionin infused directly into plasma had little effect on milk yield or composition. 26~ Again in goats, the effect was somewhat different and the amounts of only the C~8 fatty acids in milk fat were decreased by propionate infusion, an effect that might have been causally related to the diminished concentration of the free fatty acid fraction that was found in plasma. ~5'1°4 More detailed discussion of the effects of elevated rumen propionate levels on milk fat synthesis is available in Section IV.C (the low milk fat syndrome). There do not appear to have been any systematic studies of the effects of supplementation of the diets of ruminant animals with caproic, caprylic and capric acids on milk yield and composition. Tricaproin, tricaprylin, tripelargonin and tricaprin infused directly into the plasma of lactating cows over 2-day periods produced increases in milk yield proportional to the chain-length of the infused fatty acid. 26~ Considerable incorporation of these fatty acids into the milk fat were observed and some chain-elongation appeared to take place but the effects on the rate of secretion of other milk fatty acids tended to be minor, presumably because there was no effect on volatile fatty acid production in the rumen. Triundecanoin fed to lactating cows at a rate of 966g per da~ over 5 days caused a marked drop in feed consumption and a greatly reduced milk fat yield with small amounts only of undecanoic acid entering the milk fat. 59 Although the undecanoic acid appeared to be extensively metabolized in the rumen, there was no increase in the concentration of propionic acid. When triundecanoin was fed in a form protected against ruminal hydrolysis, on the other hand, there was no drop in feed consumption or in the yield of milk fat and somewhat more undecanoic acid, together with tridecanoic and pentadecanoic acids, was found in the milk fat. It would appear, therefore, that undecanoic acid exerts its effect on mammary lipid metabolism indirectly by influencing rumen metabolism. Lauric acid, either in pure form or in a natural esterified form as in coconut oil, where it comprises approximately 50%0 of the total fatty acids (together with myristic acid, up to 20~o; caprylic acid, 6-90/0; and capric acid 6-8~o) has been fed as a dietary supplement to cows in a number of experiments. Such supplements fed at low levels

Effects of diet on lipid composition

265

TABLE 10. Milk Lipids--The Effect of Feeding Diets Supplemented with Lauric, Myristic, Palmitic and Stearic Acids to Lactating Cows on Milk Fatty Acid Composition (Weight Percent of the Total Fatty Acids) 246 Fatty acid supplement Milk fatty acids 4:0-8:0 10:0 12:0 14:0 14:1 16:0 16: I 18:0 18:1 18:2 + 18:3

Unsupplemented

14:0

16:0

18:0

12:0 ~

7.0 0.5 2.1 11.4 0.5 38.7 1.3 10.I 21.3 2.9

5.3 0.3 1.6 31.6 2.4 31.8 2.1 5.5 15.3 1.8

5.6 0.1 0.9 6.3 0.4 60.7 3.5 4.3 14.2 1.7

6.9 0.5 1.2 9.2 0.2 27.7 1.2 18.7 30.1 1.4

5.0 0.4 11.4 13.5 36.3 5.3 18.5 3.2

~Data from a separate experiment with a single cow.

(up to 4~o of the concentrate fraction of the diet) especially when fed for short periods (less than 14 days) had little effect on milk fat yield 162'214'246'25a'265'273 but, as the amount of lauric acid secreted in the milk fat increased markedly, there was a net diminution in the rate of synthesis of all other fatty acids. At higher levels (5-10~o of the concentrates) of coconut oil supplementation of the diets of cows and goats, especially when fed for 22 days or longer, there was a pronounced reduction in milk fat yield; yields of lauric and myristic acids in milk fat increased but there was an appreciable drop in the rate of secretion of all other fatty acids with the possible exception of the Cls components, sS'254,25s'273 The proportion of lauric acid in milk was, therefore, higher in relation to those of the other fatty acids as is illustrated by the results in Table 10 which were taken from a representative experiment. 246 The amount of lauric acid found in the milk fat was directly related to the amount in the diet. 25s These effects on milk fat yield appeared to be a consequence of the diminished availability of the required precursors for fatty acid synthesis in mammary gland as the concentrations of acetic and butyric acids in the rumen were decreased by dietary lauric acid while that of propionic acid was increased. 214'246'254 (It is assumed that these steady-state concentrations reflect the rate of production of the volatile fatty acids.) When coconut oil was fed in a form protected against hydrolysis in the rumen by casein-formaldehyde treatment, no changes in the concentrations of the rumen volatile fatty acids were noted and there was no effect on the rate of fatty acid synthesis in the mammary gland although lauric acid was still esterified in large amounts; 254 there was, accordingly, a net increase in the yield of milk fat. 15'254 Similar effects were observed when trilaurin and trimyristin were infused directly into the plasma of lactating cows over a 2-day period. 267 Myristic acid (95~o pure), fed to cows in amounts corresponding to 10~o by weight of the concentrate fraction in the diet for 20 days, had no effect on total milk fat yield although there again appeared to be a drop in the rate of fatty acid synthesis by the mammary gland as the proportions of myristic, myristoleic and palmitoleic acids in milk fat increased in relation to those of all other components. 246 No chain elongation of lauric or myristic acids in mammary gland was apparent. 246 Some effects of dietary supplements of coconut oil, fed to cows in native form or protected against hydrolysis in the rumen, on the yield and composition of the lipids of the milk fat globule membrane have been observed.5 Palmitic acid fed as a dietary supplement to lactating cows over 20-35-day periods at a level of 10% by weight of the concentrate fraction in the ration, produced significant improvements in the daily yield of milk fat and in the yields (and, therefore, the relative proportions) of palmitic and palmitoleic acids in milk fat in comparison to those of control animals (see Table 10) 177'242'246 and at the same time, there were slightly diminished yields of some of the medium chain-length fatty acids. The additional palmi-

266

W.W. Christie

toleic acid found was presumably formed by desaturation of palmitic acid, a process that is known to occur in the m a m m a r y gland although at only 20°,4 of tile rate for stearic acid. 25 The increased yield of palmitic acid in milk fat could be correlated with a similar increase in the relative proportion of this component in the triglyceride and free fatty acid fractions of the plasma. ~7° In these experiments, the dietary supplement appeared to have only minor effects on the concentrations of individual volatile fatt~ acids in the rumen; 177'24~' the small drop in fatty acid synthesis, therefore, may not have been related to changes in the concentrations of the short-chain precursors, A possible explanation in this instance is that the exogenous fatty acids inhibited the rate-limiting enzyme in fatty acid synthesis, i.e. acetyl-CoA carboxylase. I~'L~ Similar effects, in general, were obtained in feeding palmitic acid-rich concentrates to lactating cows 2°'-'1"245 and conflicting results for minor changes in the proportions of stearic and oleic acids in milk fat may helve been due to differences ill the fat compositions of the basal diets. ~7~'24~' Butter prepared from milk fat with such a high palmitic acid content was relatively hard and high melting. 246 When dietary supplements of stearic acid (5 10% by weight of the concentrate fraction of the diet) were fed to lactating cows or goats for up to 35 days, there were also increased daily yields of milk fat. s s,ss.l o9.~ 7v,2,~s,_,4~,,2~7 Yields of both stearic and oleic acids increased appreciably whereas those of most of the medium chain-length fatty acids tended to decrease. It appeared that synthesis of fatty acids de not:o in the mammary gland was inhibited slightly although only minor changes in the concentrations of potential precursors, i.e. the volatile fatty acids in the rumen, were observed. 17~':'~5 Again, inhibition of acetyl-CoA carboxylase might be implicated. The additional oleic acid that was found in the milk fat of animals receiving dietary stearic acid supplements was presumably produced by desaturation of oleic acid in the m a m m a r y gland itself (see M o o r e and Christie ~~6a). ~4"25'30"131"141 Analysis of plasma lipids in cows receiving a dietary stearic acid supplement appeared Io confirm that this was so since the plasma triglyceride fraction contained similar amounts of oleic acid as was found in the corresponding fraction from control animals, although the former contained much more stearic acid. ~7° Under normal circumstances, it appears that the oleic acid content of milk fat is linearly related to the stearic acid content. 2~ All the newly-synthesized oleic acid was found to be incorporated into one of the primary positions of the triglyceride molecule by microsomal preparations from mammarx, gland and Kinsella ~32 has suggested that position sn-3 specifically might be involved. i.e. the last position to be acylated during triglyceride biosynthesis by the >glycerophosphate pathway, and that stearic acid desaturation might be a regulatory step in milk triglyceride biosynthesis. Triglycerides of medium chain-length fatty acids infused into plasma appeared to inhibit stearic acid desaturation, e~,v Similarly, when cyclopropene fatty acids, protected against biohydrogenation in the rumen by formaldehyde-casein treatment, were fed to lactating cows, "~5 there were substantial increases in the proportion of stearic acid in milk fat as was anticipated as cyclopropene fatty acids were known to be potent inhibitors of stearic acid desaturation (reviewed by Christie3O). When the cyclopropene fatty acids were not protected in this way, there was no such effect, presumably because they were hydrogenated in the tureen. When dietary supplements of oleic acid were ted to cows a t levels of 5 or 101~i, b\ ' weight of the concentrate fraction, the daily yield of milk fal fell. particularly in tile animals receiving the higher amount of fat in the diet. 24-s At the lower level of supplementation, only the yields and relative percentages of stearic and oleic acids in the milk fat tended to increase and at the higher level of supplementation, only those of oleic acid increased; at the same time, the yields and relative percentages of all the other fatty acids of milk fat tended to decrease. It seems probable that the dietary oleic acid is hydrogenated to stearic acid in the rumen and that this is subsequently' desaturated back to oleic acid in the m a m m a r y gland but no direct experimental.confirmation appears to be available. The reduced secretion of fatty acids was probably due to diminished availability of short-chain precursors for fatty acid synthesis, as the con-

Effects of diet on lipid composition

267

centration of acetate in the rumen was lowered by the treatments whereas that of propionate was increased. Triolein infused directly into the plasma increased the yield of milk fat mainly by increasing the yield of oleic acid in the fat; there appeared to be little effect on fatty acid synthesis in this instance. 267 Elaidic acid infused into the abomasum of lactating cows had no effect on milk fat yield or on plasma lipid composition.215 One of the [east expensive and most abundant sources of comparatively saturated fatty acids suitable for animal consumption is "tallow". Generally this consists of fat rendered from the carcasses of cattle or sheep during meat processing and contains high proportions of stearic and octadecenoic acids, including appreciable amounts of trans-isomers, and palmitic acid. Occasionally pig fat or "lard", which contains up to 10% linoleic acid and very little trans-fatty acids, may be admixed with tallow. Another potential source of saturated and monoenoic Cla fatty acids is partially hydrogenated vegetable oils, while partially hydrogenated marine oils contain C20 and C22 components also; again, oils that have been subjected to catalytic hydrogenation tend to contain high proportions of trans-isomers. In a number of experiments in which high levels of tallow or hydrogenated vegetable oil (up to 10% of the concentrate fraction of the ration) were fed to lactating cows, significant improvements in the daily yield of milk fat were obtained 21"31"2°4"243 but in others, no such effect was observed. 2'46'166'259 No definitive explanation for the discrepancies is available but such factors as the nature and composition of the basal diet, especially that of the roughage component, the stage of lactation of the experimental animals or climate might be involved. In only one experiment ~5° was there no apparent effect of dietary supplementation with tallow on the fatty acid composition of the milk fat, while in all others, the yields and relative percentages of the 18:0 and 18:1 fatty acids were increased and those of medium-chain fatty acids tended to decrease. 2°'166'243'259 Storry et al. 259 for example, fed tallow at various levels to lactating cows and showed that the yields of 18:0 and 18:1 fatty acids in milk fat were positively correlated with the daily intake of these components. On the other hand, the yields of 14:0 and 16:0 fatty acids in milk fat were negatively correlated with the daily intake, i.e. the synthesis of these fatty acids in mammary gland was greatly depressed. Of the short-chain fatty acids in milk fat, only butyric acid secretion was not diminished by the treatments. The composition of milk fatty acids, therefore, represented a balance between increased uptake of exogenous fatty acids by the mammary gland and decreased synthesis of fatty acids in the gland. Approximately 10% of the 18:1 fatty acids in milk fat from lactating cows receiving tallow supplements were found to be trans-isomers. 2° The tallow supplements had no significant effects on the concentrations of the volatile fatty acids in the rumen 259 so that the reduced synthesis of short- and medium-chain fatty acids did not appear to be a consequence of diminished availability of the precursors for fatty acid synthesis, suggesting again perhaps that some inhibition of the enzymes of the fatty acid synthetase by elevated levels of exogenous fatty acids was occurring. Results obtained by feeding supplements of this nature, protected against hydrolysis and hydrogenation in the rumen by casein-formaldehyde treatment, to lactating cows were also somewhat contradictory. For example, when protected lard supplements were fed at three levels, yield of milk fat was increased 3 whereas feeding protected hydrogenated soybean oil depressed milk fat yield. ~5 Protected tallow fed at two levels 7~ produced increases in milk fat yields but the amounts of only the 4:0, 16:1, 18:0 and 18:1 fatty acids secreted increased while the yields of all the other components decreased, i.e. the results were analogous to those obtained when tallow was fed in an unprotected form. On the other hand, when dietary supplementations with protected and unprotected tallow were compared in a single experiment, there was a much greater stimulation of milk fat production with the former in comparison to the latter, although the magnitude of the effect diminished as lactation progressed. 297 The fatty acid compositions of the milks from the two treatments did not differ significantly from each other but

268

W.W. Christie

both contained much more oleic acid than was found with control cows that did not receive the supplements. Dietary supplements of hydrogenated marine fats were found to have either no effect s: or to increase 27~ milk fat yield; appreciable amounts of C20 and C22 saturated and monoenoic (including trans-isomers) fatty acids appeared in the milk fat but there were diminished yields of the medium chain-length fatty acids.

3. Polyunsaturated Fatty Acids o[" Veyetable Oils There have been innumerable experiments over the last fifty years in which the diets of lactating ruminants have been supplemented with vegetable oils, such as cottonseed. soybean, safflower, corn (maize) or linseed oils, that contain polyunsaturated fatty acids, principally linoleic and linolenic acids. The objective in most instances, expecially in more recent years, has simply been to raise the energy content or density of the diet, as it was realized that the fatty acid composition of the milk fat could not be readily modified by such means because of the extensive biohydrogenation that occurred in the rumen (see H a r f o o t ~ b ) and because of the selective esterification of dietary fatty acids that escape this process to specific plasma lipid fractions that do not supply significant amounts of fatty acids to the mammary gland (see Section II.C above). Subsequently, methods of protecting the oils from hydrolysis and hydrogenation in the rumen were developed and these permitted some manipulation of the composition of the milk fat. At the same time. such protected diets prevented any potentially inhibitory effects of polyunsaturated fatty acids on rumen microorganisms and on their production of essential metabolites (see Section I above). Comparisons of results from experiments in which native vegetable oils were fed as dietary supplements to lactating cows were rendered difficult by the fact that frequently insufficient numbers of animals were assigned to specific treatments to permit proper statistical evaluation. In addition, variations in the level of feeding, in the nature of the basal diet and in the duration of the trials must be taken into account. In most comparatively long-term (4 6 week periods on each treatment) feeding trials and in some of shorter duration in which vegetable oil supplements were added to the diets, there was either no significant effect on milk fat yield or it was depressed relative to that of control animals receiving no such dietary supplement. 2.31,100,149.166.182,20t'24"3"244'247'28e' On the other hand, in some experiments in which these supplements were fed for shorter periods (2-3 weeks), some increases in milk fat yield were found. 2~'55'~4°'~5s'2~7"2°5 The length of the feeding period did appear to be important as Steele and Moore 2"~3 found that when dietary supplements of cottonseed oil (10°o of the concentrate fractions) were fed to lactating cows, the milk fat yield was increased during the first 8 days of the 4-week treatment period but was markedly depressed during the last 4 days. It seems possible that the nature of the populations of rumen microorganisms changes with time under the influence of comparatively large amounts of dietary unsaturated fatty acids and that this leads to changes in the availability of essential metabolites to the host animal. The physical state in which the oil is supplied may also be important as it was found in one experiment -~4" that soybean oil itself depressed milk fat yield considerably although the yield was actually increased when an equivalent amount of oil was fed in the form of intact soybeans. Fortunately, there is near unanimity on the effects of dietary supplements of unsaturated fatty acids on the composition of milk fat. Some results from a representative experiment 243 are listed in Table 11. In virtually all experiments, it was shown that the relative proportions of 18:0 and 18:1 fatty acids in milk fat tended to increase markedly whereas those of the medium chain-length fatty acids tended to decrease relative to the corresponding values from control animals; there were rarely any changes in the proportions of linoleic and linolenic acids. 20,31,4"6,55'100"138"140"150"158"166"182'201'205"243"244'248'257'265"282 These results were

Effects of diet on lipid composition

269

TABLE 11. Milk L i p i d s - - F a t t y Acid Composition (Weight Percent of the Total) and Daily Yield" (g) of Individual Fatty Acids in Milk Fat of Cows Receiving Dietary Supplements of Cottonseed Oil or Control (Unsupplemented) Diets 243 Weight percentage

Daily yield

Fatty acids

Control

Cottonseed oil supplement

Control

Cottonseed oil supplement

4: 0 - 8 : 0 10:0 12:0 14:0 16: 0 16:1 18:0 18:1 18:2 + 18:3

8.1 2.2 2.5 12.5 37.5 3.1 8.1 17.8 2.3

79 0.6 1.4 7.8 h 33.5" 2.2 12.8 h 26.7 h 2.9

53.4 14.4 16.8 82.7 248.0 20.6 53.1 117.0 14.8

47.8 ~ 3.8 h 8.6 ¢ 47.6 h 203.0 h 13.5 77.8 b 162.0 h 17.6 h

~Measurements during the last 4 days of a 4-week period. h'~Significantly different (P < 0.05, P < 0.01, respectively), from the values obtained with the unsupplemented ration.

the consequence of biohydrogenation,in the rumen and of the selective plasma transport system described earlier (Section II.C). Much of the stearic acid that was absorbed was desaturated to oleic acid in the mammary gland (see Section IV.B.2 above) but a high proportion (up to 25Y/o) of the component designated as a 18:1 fatty acid may have consisted of trans-isomers 2°'138"~4°'244'248 that were produced as by-products of the biohydrogenation process. In some experiments, the proportions of linoleic acid that appeared in the milk fat were greater than in others but this may have been due to differences in the biohydrogenation capacity of different populations of rumen microorganisms. Similarly, the daily yield of 18:0 and 18:1 fatty acids tended to increase markedly when dietary supplements of vegetable oils were fed to lactating cows (see also Table 11).20"158'243'24"8'257'299 At the same time, the yields of most of the short and medium chain-length fatty acids were diminished, apparently as a consequence of reduced synthesis de novo within the mammary gland, although it appeared that increased amounts of butyric acid might have been synthesized.248'257 The recorded effects of these supplements on volatile fatty acid concentrations in the rumen were generally negligible 31'158'~82 although in two experiments, 247'286 small but significant increases in propionate concentration relative to acetate concentration were found. It would, therefore, appear that the inhibition of fatty acid synthesis in mammary gland may have been due in part to reduced availability of acetate as substrate but in greater measure to inhibition of one of the enzymes of the fatty acid synthetase, probably acetyl-CoA carboxylase. In recent years, there has been a great deal of interest in raising the polyunsaturated fatty acid levels in milk fat and dairy products by means of feeding dietary supplements of vegetable oils protected against hydrolysis and biohydrogenation in the rumen (see Section I above), for example, by treatment with casein and formaldehyde. This interest has provided the stimulus for a number of other reviews on the topic. 106'107'126'224'253 In most of the feeding trials in which dietary supplements of vegetable oils prepared in this way were fed to lactating cows, together with earlier experiments in which oils were infused into the abomasum or directly into the bloodstream, the daily yield of milk fat was increased considerably, sometimes by as much as 259/o over that of control animals receiving an unsupplemented diet. 3'13,15'27,38,1°°,134,158,197,2°5,26°,262,3°° (In a few experiments only, no significant change was observed. 99'1°1'215,272) This increase was a consequence of additional amounts of linoleic acid being taken up by the mammary gland as up to 35~ of the total fatty acids in milk fat consisted of this component, the actual proportion being dependent on the amount of protected lipid fed, the duration of the feeding trial and the efficiency of the protection proce-

270

W . W . Christie TABLE 12. Milk Lipids---Fatty Acid C o m p o sition (Weight Percent of the Total) on Feeding Formaldehyde-protected Casein (Control) and Foimaldehyde Casein Protected Safflower Oil to Lactating Cows )°~ Fatty acid

Control

Protected lipid

4:0 6:0 8:0 10:0 12:0 14:0 16:0 t6:1 18:0 18:1 18:2 18:3

3.6 3.3 1.3 2.5 2.9 10.4 30.4 1.9 12.1 24.2 2.6 1.7

1.6 2.2 0.9 2.4 2.7 8.3 15.6 1.1 11.5 24.2 24.6 2.4

Some results from a representative experiment ~°~ are listed in Table 12 to illustrate the point. In absolute terms, the transfer of protected linoleic acid from the diet to the milk fat was as much as 40~. As the linoleic acid concentration of the milk fat increased, the relative proportions of the medium chain-length (I0:0, 12:0, 14:0 and 16:0) fatty acids were diminished. The 18:0 and 18: I fatty acids either did not change in concentration or increased slightly, probably because some of the dietary linoleate was not adequately protected and was hydrolyzed to the free acid and hydrogenated in the rumen. In view of the many feeding trials that have been performed with protected unsaturated lipids, the effects of such compounds on lipogenesis in the mammary gland and on the daily yields of individual fatty acids, other than linoleic, in the milk fat were surprisingly poorly documented. It did appear, however, as might be anticipated that marked inhibition of the synthesis of medium chain-length fatty acids occurred. Direct inhibition of the fatty acid synthetase is possible as has been postulated on a number of occasions above. Gooden and Lascelles, I°I on the other hand, found a substantial decrease in the arterial concentration of acetate and in the arteriovenous difference when such protected diets were fed. The decreased fatty acid synthesis may then have been simply, due to a diminished availability of substrate. In milk fat from cows receiving diets rich in protected linoleic acid, increased amounts of this component were found in all three positions of the triglycerides although the greatest amounts were found in positions sn-2 and sn-3,16~ The compositions of the lipids of the milk globule membranes from such milk have also been determined. 23s'237 No change in the cholesterol content of the milk was observed. 29:'3°° There are a number of technical problems associated with the commercial preparation of dairy products from milk containing high levels of linoleic acid of which the most important is probably prevention of autoxidation. 49,72.99't34'z43,29s'296 These problems have been discussed in several recent reviews. 1°6,x°7,l)l'128,t29 dure.3.13.15,17,27.28,38.4o,72,99

i01.134,138,1

~ ~ ~ ~'~ "~ ~ ~ ~8.-05,-15.~_5,..0,_60._7.._98.300

4. Oligounsaturated Fatty Acids of Cod-liver Oil

It has long been known (see Hilditch and Williams 116) that feeding dietary supplements of cod-liver oil to lactating cows depresses milk fat yield and this has been confirmed by a number of recent studies. 32'2°3'256'26°'273'274"286 It appeared that the daily yields of the C4-C~6 fatty acids, i.e. those derived mainly by synthesis de novo in the mammary gland, and of the CIs fatty acids, i.e. those taken up from the plasma and which were derived originally from the diet, were reduced; only small amounts of C20 and C22 oligounsaturated components from the oil were found in the milk fat.32,2o3,256,274

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Although the nature of the effects is well understood, the reasons for them are not altogether clear. One major factor involved in the reduced rate of fatty acid synthesis was that the administration of cod-liver oil produced a change in the fermentation pattern in the rumen reducing the concentrations of acetate and occasionally butyrate while increasing the concentration of propionate, 32"t83"23°'273'274 i.e. the availability of the primary substrate for the fatty acid synthetase in mammary gland was reduced. There did not appear to be any appreciable inhibition of mammary gland fatty acid synthesis directly by cod-liver oil fatty acids as when the oil was administered in ways that by-passed the rumen microflora,2°*'~56'2~° the daily yields of the C4-C16 fatty acids were close to those of control animals receiving an unsupplemented diet. Administration of cod-liver oil to ruminants also apparently reduced the rate of synthesis of long-chain fatty acids de novo by rumen microorganisms so that diminished amounts of fatty acids from this source would be available to the mammary gland. 256 For some time, it was thought that the oligounsaturated fatty acids of cod-liver oil incorporated into the plasma triglyceride fraction might be resistant to hydrolysis by lipoprotein lipase or that such acids in an unesterified form might inhibit lipoprotein lipase reducing the availability of fatty acids to the mammary gland, but these hypotheses no longer appear to be tenable. Although cod-liver oil emulsions were apparently hydro° lyzed more slowly than soybean oil dispersions by lipoprotein lipase in vitro, small amounts only of oligounsaturated fatty acids were found in the plasma triglyceride fraction of cows receiving cod-liver supplements so that this fraction could not in any case be a major supplier of such components to the mammary gland, a2 Others 17~ have reported that bovine milk lipoprotein lipase hydrolyzed oligounsaturated fatty acids as readily as Cls polyunsaturated fatty acids from triglycerides and that any apparent specificity of the enzyme simply reflected the fact that the oligounsaturated components tended to be concentrated in positions sn-2 and sn-3 of natural triglycerides while the fatty acids of position sn-1 were released first by the enzyme. Oligounsaturated fatty acids that would be available for uptake by the mammary gland were concentrated in the free fatty acid fraction of plasma 32 (see also Section II.C above) but these did not appear to inhibit lipoprotein lipase as high density lipoproteins from cows receiving cod-liver oil supplements were equally effective as activators of lipoprotein lipase as were the corresponding lipoproteins from cows receiving hydrogenated cod-liver oil supplements. It has been suggested more recently a2,26s that unesterified oligounsaturated fatty acids in plasma may inhibit the uptake of long-chain fatty acids by the mammary gland capillary endothelial cell by some as yet unknown mechanism. C. The Low Milk Fat Syndrome

When diets containing a low proportion of roughage are fed to lactating cows, marked falls in the content of the fat in milk and in the overall yield of milk fat can result; the phenomenon is often described as the low milk fat syndrome. 18'31"52'78'79'85'133'148't65'206'208'231'243'244'255'262-264'266'276 In the U.K., the problem is most often seen during the early spring when the diets of the cows are changed from a mixture of hay and concentrates to one of fresh grass. In the U.S.A., it is observed when, for reasons concerned with climate, husbandry or economy, diets containing a high proportion of concentrates relative to roughage are fed. The magnitude of the effect is influenced by a number of factors including the physical form of the diet (e.g. whether it is pelleted, ground or flaked), the stage of lactation or the physical condition of the animal and the level and frequency of feeding. Reductions in milk fat yield of 50-60~ have been recorded and, in some experiments, changes in fatty acid composition have been observed with higher proportions of unsaturated fatty acids, especially of trans-monoenoic components, being found in the milk fat. of cows on lowroughage diets. 263 The reductions in milk fat yield brought about by feeding dietary supplements of unsaturated oils to cows are sometimes considered to be a part of

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the low milk fat syndrome but this aspect was discussed in the previous section of this article. The syndrome obviously has considerable economic importance and has been the subject of a number of r e v i e w s . 8 ' 2 4 ' 5 1 ' 7 7 ' 2 5 1 ' 2 5 2 ' 2 8 3 A number of hypotheses have been put forward to explain the syndrome that involve a reduction in the availability of some essential substrate required for fatty acid synthesis in the mammary gland or a more general alteration to the normal lipid metabolism of the animal and it is now apparent that several factors are involved. For example, it is well documented that the molar ratio of acetate to propionate in the rumen of cows on low-roughage diets is greatly increased 1s'203'264 and it has been suggested that diminished availability of acetate would lead to a reduction in fatty acid synthesis in the mammary gland. On the other hand, Davis 5° reported that acetate production was not affected by low-roughage regimes and that the acetate concentration in the rumen was not proportional to the production rate. Attempts to increase the amount of acetate available to the mammary gland by administering acetate to cows on lowroughage diets yielded results that were highly variable 51 although, in similar experiments with cows on more normal diets, improved milk fat yields were demonstrated (see previous section). Similarly, studies of the effect of low-roughage diets on plasma fl-hydroxybutyrate levels have given inconclusive results. 51 However, reduced mammary gland uptake of acetate and fl-hydroxybutyrate does appear to have been demonstrated by direct measurement in cows on milk fat-depressing diets. 2~'26 It is also well documented 5~ that rumen propionate levels are considerably elevated when low-roughage diets are fed: indeed, McCullough ~$3 showed that there was an excellent correlation between rumen propionate levels and the degree of milk fat depression. It has been suggested that elevated propionate levels might simply diminish the availability of fl-hydroxybutyrate for fatty acid synthesis 284 but a more likely explanation ~5~'~Sz is that increased propionate concentrations in the rumen lead to increased lactate and glucose production which in turn stimulate insulin production reducing the rate of release of free fatty acids from adipose tissue: accordingly, there would be a diminished availability of preformed long-chain fatty acids for milk fat synthesis. Although this hypothesis appears to be plausible, it has not proved easy to fully verify it experimentally. 5t However, when rumen propionate levels were artificially elevated, there was no corresponding increase in propionate concentration in the plasma, which would suggest that the effects on milk fat production were mediated through some metabolite or other. It does appear to have been established that mobilization of free fatty acids from adipose tissue is reduced in cows producing low fat milk. 8 Also, there is a considerable body of evidence to suggest that there is increased synthesis and esterification of fatty acids in adipose tissue of cows fed low-roughage d i e t s . 12"19"22"35"108"192"193'304 These effects, which are discussed in greater detail elsewhere 28s~ could also be a possible consequence of increased plasma insulin levels and might indirectly limit mammary lipid synthesis by making use of acetate or long-chain fatty acids that would otherwise by available to the mammary gland. The availability of preformed long-chain fatty acids to the mammary gland is undoubtedly an important factor in the low milk fat syndrome. When appreciable amounts of long-chain fatty acids were administered to cows on low-roughage milk-depressing diets in a manner such that the rumen was effectively by-passed, for example by infusing cottonseed or soybean oils into the plasma 26°'262 or by feeding tallow protected against ruminal hydrolysis by casein-formaldehyde treatmentf155 sufficient fatty acid was made available to both the adipose tissue and mammary gland and there were significant improvements in the milk fat yield.

(Received 11 April 19781 V. R E F E R E N C E S 1. ACKERSON, B. A., JOHNSON, R. R. and HENDRICKSON, R. L. J. Nutr. 106, 1383 1390 (19761. 2. ADAMS, H. P., BOITMAN V. R., LESPERANCE, A. L. and BRYANT. J. M. d. Dairy Sci. 52. 169-171 (1969).

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