Factors Affecting Conjugated Linoleic Acid Content in Milk and Meat

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1 Critical Reviews in Food Science and Nutrition, 45: (2005) Copyright C Taylor and Francis Inc. ISSN: DOI: / Factors Affecting Conjugated Linoleic Acid Content in Milk and Meat TILAK R. DHIMAN, SEUNG-HEE NAM, and AMY L. URE Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, UT , USA Conjugated linoleic acid (CLA) has been recently studied mainly because of its potential in protecting against cancer, atherogenesis, and diabetes. Conjugated linoleic acid (CLA) is a collective term for a series of conjugated dienoic positional and geometrical isomers of linoleic acid, which are found in relative abundance in milk and tissue fat of ruminants compared with other foods. The cis-9, trans-11 isomer is the principle dietary form of CLA found in ruminant products and is produced by partial ruminal biohydrogenation of linoleic acid or by endogenous synthesis in the tissues themselves. The CLA content in milk and meat is affected by several factors, such as animal s breed, age, diet, and management factors related to feed supplements affecting the diet. Conjugated linoleic acid in milk or meat has been shown to be a stable compound under normal cooking and storage conditions. Total CLA content in milk or dairy products ranges from 0.34 to 1.07% of total fat. Total CLA content in raw or processed beef ranges from 0.12 to 0.68% of total fat. It is currently estimated that the average adult consumes only one third to one half of the amount of CLA that has been shown to reduce cancer in animal studies. For this reason, increasing the CLA contents of milk and meat has the potential to raise the nutritive and therapeutic values of dairy products and meat. Keywords cancer, conjugated linoleic acid, fat, food, meat, milk, ruminant INTRODUCTION Utilizing the diet as a means of controlling and reducing the incidence of cancer in humans has received considerable attention. Firm evidence of its value, however, is sparse, and very little new evidence has been obtained in the past decade. There is a growing interest in natural nutrients and non-nutrients that are present in foods that may have health benefits for humans. One of these nutrients is conjugated linoleic acid (CLA). Conjugated linoleic acid occurs naturally in many foods; however, principle dietary sources are dairy products and other foods derived from ruminants. 1 Over 60 years ago, Bank and Hilditch 2 showed that feeding liberal amounts of highly unsaturated oils to steers over a period of 260 days had no effect on the level of unsaturation of body fat. Later, Shortland et al. 3 observed that although the main dietary fat in pasture-fed animals is linolenic acid (C 18:3 ), it is only present in trace amounts in the depot fat of rumi- Approved as journal paper number 7573 of the Utah Agricultural Experiment Station, Utah State University, Logan, The use of trade names in this publication does not imply endorsement by the Utah Agricultural Experiment Station, Utah State University of products named, nor criticism of similar ones not mentioned. Address correspondence to Tilak R. Dhiman, Ph.D., Department of Animal, Dairy, and Veterinary Sciences, Utah State University, Logan, UT , USA. trdhiman@cc.usu.edu 463 nants. The first evidence of ruminal biohydrogenation of dietary lipids was provided by Reiser 4, Hartman et al., 5 and Shortland et al. 6 It was also established that the process of biohydrogenation in the rumen was incomplete, and that unsaturated fatty acids were saturated by ruminal microoganisms. 5 The presence of conjugated unsaturated fatty acids in milk was first observed by Booth et al., 7 who reported that milk fat from cows grazing pasture in the summer showed an increased absorption in the ultraviolet region (230 nm) as compared to milk fat produced by the same cows during the winter months. During those years, it was a common practice for cows to graze during the summer months and receive dry forage in the winter months. Forty years later, Parodi 8 isolated cis-9, trans-11 C 18:2 (c9, t11 CLA) from milk fat and suggested that fatty acids with conjugated unsaturation are not normally part of a cow s diet, but that they appear in milk as a result of ruminal biohydrogenation of lipids. Ten years later, Ha et al. 9 isolated CLA from grilled ground beef and showed that synthetically prepared CLA inhibited the initiation of mouse skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene. Since then, there have been numerous research studies conducted in an attempt to understand the synthesis of CLA, its mechanisms of action, and its content in natural foods. Conjugated linoleic acid (mixtures of cis-9, trans-11 and trans-10, cis-12 isomers) has been shown to have anticancer

2 464 T. R. DHIMAN ET AL. properties in various studies in animal models The mechanisms whereby this occurs are not known, but some theories are that CLA reduces cell proliferation, alters various components of the cell cycle, and induces apoptosis. 23 In several human cancer studies, an inverse association was found between the level of CLA in the diet and the risk of developing cancer in breast adipose tissue Studies conducted with mice, chickens, and pigs have suggested a possible role of CLA (mainly the trans-10, cis-12 isomer) in decreasing body fat and increasing lean body mass A human-related study has suggested that CLA increases body mass without increasing body fat. 34 Several studies indicate that CLA may enhance immune function Conjugated linoleic acid has also been found to have antidiabetic and antiatherosclerotic properties in animal models Presently, whole milk contains an average of 3.5% fat and 0.5% of the total fat is CLA. For humans, one serving of whole milk (227 ml) and one serving of cheese (30 g) daily can provide 90 mg of CLA. Using 600 g as a value for the daily food intake of the average adult, 90 mg of CLA would represent 0.015% of the diet. Unfortunately, this figure only amounts to 25% of the lowest effective dose for reducing the incidence of cancer in laboratory rats; this was reported by Ip et al. 11 Ritzenthaler et al. 46 estimated the actual average CLA intake of humans to be 150 mg/d. Again assuming a daily food intake of 600 g, this level of CLA still only amounts to 0.025% of the diet. For this reason, increasing the CLA contents of milk and meat has the potential to raise the nutritive and therapeutic values of meat and dairy products. The intake of CLA in the human diet can be increased either by increasing the consumption of foods of ruminant origin, or by increasing the CLA content of milk and meat. As the latter approach is more practical, several research studies have been conducted during the last decade in an effort to enhance the CLA content of milk and meat. Recently, Bauman et al. 47 published an excellent article describing the biosynthesis of CLA in ruminants. Other articles have focused on the potential health benefits of CLA. 48,49 The objective of this article is to provide only a brief overview of CLA synthesis in ruminants and then examine in detail the role of animal s diet, management, genetics and their ability to influence the CLA content of milk and meat, as well as processed products, thereby making them even better food sources for human consumption. CLA ISOMERS Conjugated linoleic acid is a collective term for a series of conjugated dienoic positional and geometrical isomers of linoleic acid (C 18:2 ). Conjugated linoleic acid isomers are found naturally in foods, especially those of ruminant origin. 1 In ruminants, CLA is synthesized by ruminal bacteria using C 18:2 or C 18:3 as the precursor. 50 Conjugated linoleic acid isomers can also be synthesized in the laboratory from C 18:2 or from sources high in C 18:2, such as sunflower, safflower, soybean, or corn oils, by a reaction involving alkaline water isomerization 51 and isomerization in propylene glycol. 52 The cis-9, trans-11 isomer is the principle dietary form of CLA exhibiting biological activity and accounts for 73 to 94% of total CLA in milk, dairy products, meat, and processed meat products of ruminant origin. 8,53 55 In recent years, biological activities have been proposed for other CLA isomers, especially trans-10, cis-12 C 18:2 29,30 (t10, c12 CLA). Throughout the rest of the text, the cis double bond will be abbreviated as c and the trans double bond as t. The structures of c9, t11 CLA, t10, c12 CLA, and C 18:2 are shown in Figure 1. A total of 17 natural CLA isomers have been found in milk, dairy products, beef, human milk, and human adipose tissue using silver ion-high performance liquid chromatography and gas chromatography-electron ionization mass spectrometry. 1,52,56 61 Identified CLA isomers are t12, t14; t11, t13; t10, t12; t9, t11; t8, t10; t7, t9; t7, c9; t6, t8; c12, t14; t11, c13; c11, t13; c10, t12; c9, t11; c8, t10; c7, t9; c9, c11; and c11, c13. Bauman et al. 62 observed that butter contained c9, t11 (76.5%) and c7, t9 (6.7%) isomers. Sehat et al. 52 identified the distribution of CLA isomers in cheese fat: c9, t11 (78 to 84%); t7, c9 plus t8, c10 (8 to 13%); t11, c13 (1 to 2%); c12, t14 (<1%); their total trans/trans isomers (5 to 9%). Recently, Fritsche et al. 61 identified the distribution of CLA isomers in beef samples and found that c9, t11 was the predominant isomer (72%), followed by the t7, c9 isomer (7.0%). Typical synthetic CLA isomer mixtures consist of c9, t11 (40.8 to 41.1%); t10, c12 (43.5 to 44.9%); t9, t11/t10, t12 (4.6 to 10.0%) isomers. 1,52 Christie et al. 51 and Fritsche 63 reported on a different synthetic CLA isomer mixture that contained c8, t10 (14%); c9, t11 (30%); t10, c12 (31%); c11, t13 (24%). Most of the aforementioned CLA isomers are present in foods in very minute amounts and are of little biological importance or have not been studied in detail. Therefore, the ensuing discussion will focus on the two predominant forms of CLA, namely the c9, t11 and t10, c12 isomers. CLA BIOSYNTHESIS Conjugated linoleic acid originates from either ruminal biohydrogenation of C 18:2 and C 18:3 or from endogenous synthesis in tissues as shown in Figure 2. Ruminally, CLA is produced as an intermediate product during the biohydrogenation of dietary C 18:2 or C 18:3 to stearic acid (C 18:0 ). Endogenously, CLA is synthesized from t11, C 18:1 vaccenic acid (TVA), another intermediate of ruminal biohydrogenation, via 9 -desaturase. 47 The endogenous synthesis of CLA from TVA has been proposed as being the major pathway of CLA synthesis in lactating cows, accounting for an estimated 78% of the CLA in milk fat. 64,65 Ruminal Biohydrogenation Lipids in the ruminant diet are derived from forages, grains, and oil supplements. The lipid content in most ruminant diets

3 Figure 1 (C). LINOLEIC ACID CONTENT IN MILK AND MEAT 465 Abbreviated chemical structures of ordinary C 18:2 (linoleic acid) (A) and two major conjugated linoleic acids: c9, t11 isomer (B) and t10, c12 isomer ranges from 3 7% on a dietary dry matter (DM) basis. The fatty acid profiles of some common ruminant feeds are presented in Table 1. Most ruminant feeds of vegetable origin contain C 18:2 and/or C 18:3 as the predominant fatty acids. Feeds of animal origin, such as tallow and fish products, are likely not to be as rich in these fatty acids. Among feeds, pasture diets fed to ruminants are rich in C 18:3, representing 48 to 56% of total fatty acids (FA). Corn or grass silages are rich in C 18:2 (41% of FA) or C 18:3 (46% of FA), respectively. Alfalfa hay contains high proportions of C 18:3, while other unsaturated FA in the Figure 2 Proposed mechanism for CLA synthesis from ruminal biohydrogenation or endogenous synthesis. Conjugated linoleic acid (CLA); TVA, trans vaccenic acid; I, isomerization reaction; H, hydrogenation. Adapted and reproduced with permission. 71

4 466 T. R. DHIMAN ET AL. Table 1 Fatty acid profile of common ruminant feeds Feed C 14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2 C 18:3 Others Fatty acid, % of total reported fatty acids - Pasture Grass Clover Grass + legume Silage Grass Corn Hay alfalfa Concentrates Barley Corn Oats Wheat Byproducts Gluten meal Distillers grains Plant seeds/oils Soybean Extruded soybean 99 Extruded cottonseed 99 Sunflower Peanut Linseed Fish oil *** Animal tallow Numerical superscripts next to feed correspond to reference numbers cited in the reference section. Other fatty acids not specifically identified. Other fatty acids present in fish oil are C 20:5 (14%), C 22:6 (10%), and other PUFA (8%). Values are representative of Menhaden fish oil. green forage may become oxidized during the drying process. Most plant seeds and oils are rich in C 18:2, accounting for 53 to 69% of total FA (Table1). However, peanut oil is rich in C 18:1 (51%) and linseed oil contains an abundance of C 18:3 (51% of total FA). Fish oil contains relatively low amounts of C 18:2 and C 18:3,but is very rich in long chain polyunsaturated fatty acids (PUFA). Animal fat has a high proportion of C 18:1 (46% of total FA). When consumed by ruminants, the lipid portions of these feeds undergo two major processes in the rumen. 66,67 In the first process, esterified plant lipids or triglycerides are quickly hydrolyzed to free FA by microbial lipases. 68 In the second process, the unsaturated free FA are rapidly hydrogenated by microorganisms in the rumen to produce more highly saturated end products. The c9, t11 isomer of CLA is the first intermediate product in the biohydrogenation of C 18:2 by the enzyme linoleate isomerase (Figure 2), which is produced by the microorganism Butyrivibrio fibrisolvens 50 and other bacterial species. Part of the c9, t11 CLA is rapidly reduced to TVA or C 18:0, 69,70 becoming available for absorption in the small intestine. Similar to the biohydrogenation of C 18:2, the FA α and γ C 18:3, which are the predominant unsaturated FA in forages, also undergo isomerization and a series of reductions, ending with the formation of C 18:0 in the case of complete biohydrogenation. 71 The c9, t11 CLA and TVA often escaping complete ruminal biohydrogenation are absorbed from the intestine and incorporated into milk fat. 72,73 Studies with pure strains of ruminal bacteria have shown that most bacteria are capable of hydrogenating C 18:2 to t-c 18:1 and related isomers, but only a few have the ability to reduce C 18:2 and C 18:1 completely to C 18:0. 74 Interestingly, no single species of rumen bacteria catalyzes the complete biohydrogenation sequence. 71,75 It has been suggested that the biohydrogenation pathways are affected by several factors related to the composition of the diet consumed by the animal, including the rumen environment and the bacterial population. 73,76 78 Endogenous Synthesis It was originally assumed by the scientific community that the rumen was the primary site of origin of c9, t11 CLA in milk fat. Recently, however, it has been suggested that only a small portion of c9, t11 CLA escapes biohydrogenation in the rumen, and that the major portion of c9, t11 CLA in milk comes from endogenous synthesis in the mammary gland via a pathway involving the desaturation of TVA by the 9 -desaturase enzyme. 64,65,79 Several studies have been performed to confirm that the endogenous synthesis of CLA occurs in the mammary gland by 9 -desaturase. Trans-vaccenic acid (12.5 g/d) was infused abomasally into lactating cows for 3 d, subsequently resulting in a 40% increase in the CLA content of milk fat. 65 Using partially hydrogenated vegetable oil as a source of TVA, c9, t11 CLA production was increased by 17% in milk fat. 65 In addition, specific inhibitors of 9 -desaturase, such as sterculic oil [sterculic acid (C 19:1 ) plus malvalic acid (C 18:1 )] or sterculic acid only, were infused abomasally into lactating cows to quantify the importance of the desaturase enzyme in CLA production. Inhibition of this enzyme was reflected in the dramatic reduction in the c9, t11 CLA content of milk fat (60 71%) as well as other milk fatty acids containing a c-9 double bond, 65,80 as the 9 -desaturase enzyme is responsible for the introduction of a cis-double bond between carbons 9 and 10 of the FA. The actual estimated endogenous synthesis of c9, t11 CLA in milk fat was 64, 64 78, 65 or 80%, 81 of the total c9, t11 CLA, with different correction factors used according to the extent of enzyme inhibition by sterculic oil. There are reported species differences in the tissue distribution of 9 -desaturase. Enzyme activity and mrna abundance of 9 -desaturase are highest in the liver of rodents; however, in growing sheep and cattle, adipose tissue is found to have the highest levels In lactating ruminants, the highest activity of 9 -desaturase is found in the mammary tissue. 85 There is very little research exploring the factors that influence and regulate 9 -desaturase activity in the tissues of ruminants. More research

5 is also needed to gain a better understanding of the influence of the level of 9 -desaturase in various tissues on CLA synthesis. CLA CONTENT IN MILK The CLA content in milk fat can be affected by a cow s diet, breed, age, non-nutritive feed additives, such as ionophores, and by the use of synthetic mixtures of CLA supplements (Table 2). Among these factors, the diet is known to strongly influence the CLA content of milk and includes feedstuffs such as pasture, conserved forages, plant seed oils, cereal grains, marine oils and feeds, and animal fat. Dietary Factors Affecting Milk CLA Pasture, Conserved Forages, and Grain A number of studies have shown the positive effects of pasture-based diets on the CLA content of milk fat. Dhiman et al. 86 reported that cows grazing pasture had 500% higher CLA content in milk fat (2.21% of total FA) compared to cows fed a diet containing 50% conserved forage (hay and silages) and 50% grain (0.38% of total FA). Other researchers have also demonstrated that the CLA content of milk increased linearly as the proportion of fresh grass from pasture in the diet was increased Fresh grass contains approximately 1 to 3% FA on a DM basis, depending on the variety of grass, with the highest FA contents usually occurring in the spring and fall seasons. About to 56% of the total FA in fresh forages consists of C 18:3 (Table 1). Fresh grass supplies C 18:3 FA as a substrate for ruminal biohydrogenation. However, the abundant supply of C 18:3 from fresh grass only partly explains the large increases in CLA and TVA contents of milk fat from pasture-fed cows. Besides this, the high concentrations of soluble fiber and fermentable sugars present in fresh grass may create an environment in the rumen without lowering the ruminal ph that is favorable to the growth of the microbes responsible for CLA and TVA production. Ruminal ph is generally relatively high in cows grazing pasture compared to cows fed a combination of conserved forage and grain. Supplementing grain to cows grazing pasture decreases the CLA content of milk fat. Cows supplemented with 0, 6, or 12 kg/d of grain on pasture had 2.21, 1.43, and 0.89% CLA in milk fat, respectively. 86 Similarly, supplementing grain to cows receiving grass silage or replacing conserved grass in dairy cow diets with corn silage lowered the CLA content of milk. 88,92 Corn silage contains 20 to 40% grain on a DM basis. The addition of grain to dairy diets decreases ruminal ph. A decrease in ph will change the microbial population and affect ruminal fermentation. 77 It has been suggested that the main ruminal biohydrogenating bacteria are cellulolytic. 93,94 Reduction in ruminal ph decreases the population of cellulolytic bacteria and other microbes responsible for lipid biohydrogenation and the production of CLA and TVA. 72 LINOLEIC ACID CONTENT IN MILK AND MEAT 467 Table 2 Factors Factors affecting conjugated linoleic acid (CLA) content of milk Total CLA (% of fat) Diets Forage Freshness: Pasture Silage 86, Hay Maturity: Heading Second cutting Flowering Plant seed oils Soybean oils (3 4%) 101, Linseed oil ( %) 98, Peanut oil (5.3%) Sunflower oil (5.3%) (2.5%) Canola oil (3 3.3%) 101, Canolamide (3.3%) Safflower oil (2.5%) 100, Infusion (150 g/d) Ca-treated oils (4%): Canola, soybean, linseed Intact oil seed Raw soybean (17.5%) Full fat rapeseed ( kg) 111, Full fat extruded soybean (1.65 kg) ( %) 99,108, Full fat extruded cottonseed (12%) Whole ground flax, solin, or canola 87, Fish oils/meals Fish oils (1 3%) 114,115,118,121, ( ml/d) Fish meals (3 5.8%) 86,116, Algae (4%) Animal Fat Tallow (3 6%) 108,130, Infusion (150 g/d) Combinations of oilseeds and fish oil/meal Extruded soybean and fish oil 5.3% soybean and 1.0% fish oil 115,118, % soybean and 1.0% fish meal Sunflower/flaxseeds and fish oil 4 5% and 1.0%, respectively Management system Seasonal effects Spring 135, Summer 134,135, Winter 134,135, Fall 135, Elevation m m m Restricted Feeding Unrestricted 72,111, Restricted 72,111, Breed Holstein Pasture 90,141, (Continued on next page)

6 468 T. R. DHIMAN ET AL. Table 2 Factors affecting conjugated linoleic acid (CLA) content of milk (Continued) Factors Total CLA (% of fat) TMR 90,118,131,142, Brown Swiss Pasture TMR 118,142, Jersey Pasture 90, TMR 90,131, Ayrshire (Pasture) Guernsey (Pasture) Montebeliardes (Pasture) Normandes (Pasture) Age or lactation number Aged cow (>4 lactations) Young cow (2 4 lactations) Ionophores Monensin ( mg or 250 g) 86,92, Synthetic CLA supplements Infusing (post-ruminally) CLA-60 (150 g/d) CLA-46 (28.8 g/d) CLA-47 (248.5 g/d) CLA-88(16.3 g/d) Cis9, trans 11 (15 g/d) Trans10, cis 12 (15 g/d) Mixture g/d (pasture-fed) g/d Feeding CLA-60 (100 g/d) 153, CLA mix (30.4 g/d) Numerical superscripts next to factors correspond to reference numbers cited in the reference section. Additionally, the endogenous production of CLA in the mammary glands of pasture-fed cows cannot be excluded. The 9 - desaturase activity could differ in the mammary gland of cows grazing on pasture compared to cows fed conserved forages and grains. Forage Maturity and Preservation Forage maturity and method of preservation also seem to be important factors influencing the CLA content of milk. Cows fed immature forages have higher levels of CLA in milk than cows fed mature forage. Cows fed grass silage cut at early heading, flowering, and second cutting had 1.14, 0.48, and 0.81% CLA in milk fat, respectively. 92 The high C 18:3 content of immature grass and its low fiber content compared to mature grass probably interact to increase the production of CLA and TVA. Harvesting forage as hay decreases the proportion of C 18:3 and total FA in grass, whereas harvesting forage as silage, when carried out properly, does not. 95 The content of C 18:3 FA may decrease when forage is wilted before ensiling, or if there is undesirable fermentation during ensiling. 96,97 The amount of C 18:3 FA available to the animal as a substrate for CLA and TVA synthesis from fresh grass is much higher than that from hay or silage. Plant Oils and Seeds Feeding plant seed oils, such as sunflower, soybean, peanut, canola, and linseed increased CLA content in milk These oils are rich in C 18:2 and C 18:3 FA. Studies have found that high levels of C 18:2 and C 18:3 (such as those found in most plant seed oils) result in increased production of CLA and TVA, with the TVA potentially being additional substrate for the endogenous synthesis of c9, t11 CLA. 71,103,104 Besides directly increasing the yield of CLA and TVA, it is likely that C 18:2 inhibits the final reduction of TVA, thus increasing its accumulation in the rumen, 73 and subsequent availability to the animal. Feeding diets containing soybean oil (4%) resulted in approximately a four-fold increase in CLA content of milk fat (2.08%) over the control (0.50% of milk fat). 105 Supplementing peanut oil, sunflower oil, or linseed oil at 5.3% of dietary DM resulted in 1.33, 2.44, and 1.67% CLA in milk fat, respectively. 98 Feeding 4% canola oil to dairy goats increased CLA content to 3.2% of FA compared to 1.0% in milk fat from the control. 106 The specific FA that is most abundant in any given plant seed oil is very important in determining how much the oil will elevate the levels of CLA in milk fat. Oils rich in C 18:2 are more effective at increasing CLA in milk fat as compared to oils rich in C 18:3 or C 18:1. Dhiman et al. 105 reported that linseed oil was not as efficient at increasing CLA content in milk fat as was soybean oil. Feeding soybean oil at 4.0% of diet DM resulted in a higher CLA content of milk fat (2.08%) than supplementing linseed oil at 4.4% of diet DM (1.63% of FA). 105 Loor and Herbein 101 reported that soybean oil supplemented at 3% of the diet DM was more effective at enhancing the c9, t11 CLA content of milk fat than was canola oil supplemented at the same level (0.71 vs. 0.51% of milk fat). In other studies, dairy cows supplemented with sunflower or safflower oils that were high in c9 C 18:2 produced more CLA in milk fat than cows fed similar oils that were high in C 18:1 100,102 The reason why C 18:2 produces more CLA than C 18:1 is probably because of additional unsaturated double bonds and an extra hydrogenation step for the production of CLA and TVA in the rumen (Figure 2). In addition, it has recently been reported that c9 C 18:1 is primarily hydrogenated to C 18:0 or isomerized to various t C 18:1 isomers (mainly t-4 C 18:1 to t-10 C 18:1 ), 107 rather than directly to TVA. The feeding of ruminally protected plant oils to increase milk CLA content has yielded variable results. Dietary supplements of calcium salts of FA from canola oil, soybean oil, or linseed oil increased CLA content of milk from 0.35% in control to 1.32, 2.25, and 1.95% of milk fat, respectively. 108 The five-fold increase of CLA in this study suggests that the calcium salts of FA were not protected from ruminal biohydrogenation. Loor et al. 109 investigated the effects of feeding protected or unprotected canola oil on milk fatty acid composition. Cows were fed a control diet; a diet containing canola oil at 3.3% of dietary DM, a diet containing canolamide (made by a reaction of canola oil and ethanolamine to protect oil from ruminal biohydrogenation) at 3.3% of diet DM, or a diet containing a mixture of both canola oil and canolamide. Milk fat CLA contents were 0.5, 1.1, 0.7, and 1.0% of total FA for the four treatment groups,

7 respectively. The effects on milk CLA content suggest that the canola oil-containing diet provided sufficient substrate for CLA synthesis, and that the canolamide was partially protected from ruminal biohydrogenation. Abomasal infusion of 150 g/d of safflower oil was not successful at increasing the CLA content of milk fat. 110 This is strong evidence that ruminal biohydrogenation of C 18:2 plays a major part in the synthesis of CLA that is eventually incorporated into milk fat. Intact oil seeds are known to be less efficient than free oils at enhancing the CLA content of milk. Processing releases oil from the seeds; it then becomes available to the rumen microbes for biohydrogenation. Feeding raw seeds has little or no effect on the CLA content of milk fat, because polyunsaturated fatty acids (PUFA) in the intact seeds are relatively unavailable to the rumen microbes for biohydrogenation. 105 However, if raw seeds are processed by grinding, roasting, micronizing, flaking, or extruding, those processed seeds are effective at increasing the CLA content in milk. 87,99,108, The CLA content of milk fat was increased when cows were fed full-fat extruded soybeans (0.73% CLA) or full-fat extruded cottonseed (0.60% CLA) at 12% of diet DM compared to 0.34% CLA in cows fed low-fat soybean meal at 13.5% of diet DM. 99 The CLA content of milk fat was increased by an average of 123% when a portion of low-fat soybean meal was replaced by full-fat extruded soybeans in dairy diets. 113 Feeding 17.5% of dietary DM as extruded, micronized, or roasted soybeans to dairy cows increased the CLA content of milk fat to 0.89, 0.70, and 0.66% of total FA compared to 0.31% CLA in milk fat from cows fed raw ground soybeans. 108 Extruding full-fat soybeans at 120, 130, and 140 C increased CLA content of milk to a similar extent: CLA averaged 1.99% of milk fat for the extrusion treatments compared to 0.42% of milk fat from control cows fed raw ground soybeans. 108 There are a number of other research reports suggesting that feeding processed soybeans, canola, or flax seeds to dairy cows was more effective at increasing milk CLA content than feeding unprocessed seeds. 87,111,112,119,120 Interestingly, grazing cows receiving 460% less C 18:3 (102 g/d) from green grass had higher milk CLA content (2.21% of fat) 86 than cows fed diets containing conserved forages and grain supplemented with C 18:3 (575 g/day) through linseed oil (1.67% CLA in milk fat). 98 However, it should be acknowledged that factors other than oil supply from pasture grass are also responsible for the higher CLA content observed in grazing cows. Cows grazed on pasture or fed forage alone will produce less milk but a higher fat content than cows fed conserved forage and grains. The milk yield of grazing cows is reduced by times, but the CLA content of milk is 4 5 times higher than in milk from cows fed conserved forage and grain. Thus, the daily output of CLA from cows grazing on pasture will still be higher than from cows fed conserved forage and grain. This situation will change when cows are fed plant oils, as plant oils enhance the CLA content of milk. Therefore, caution must be taken when comparing dietary influence on CLA content and daily CLA output. LINOLEIC ACID CONTENT IN MILK AND MEAT 469 Marine Oils and Feeds The feeding of fish oil has been shown to enhance the CLA and TVA contents of milk fat, but reduced total fat content of milk. Feeding fish oil at 1.6% of the diet DM increased the CLA and TVA contents in milk fat from 0.16 and 1.03% in control to 1.55 and 7.50%, respectively. 121 Feeding diets containing 2% fish oil to dairy cows increased CLA and TVA contents by 300 and 500% in milk fat, respectively, compared to milk fat from cows fed no fish oil. 114,122 However, there was no additional increase in CLA and TVA contents when cows were fed 3% fish oil. Similar increases in CLA and TVA contents of milk fat from cows fed fish oil were confirmed by others. 108,115,118 The inclusion of marine feeds, such as fish meal or sea algae, into dairy cow diets has been shown to enhance the CLA content of milk. Including fish meal in dairy diets at 2.09 to 5.84% of diet DM increased the CLA and TVA contents of milk fat from 0.30 to 0.86% and 1.09 to 1.54% of milk fat, respectively. 86,105,123 In a similar study, feeding fish meal at 5.5% of dietary DM resulted in small increases in CLA and TVA (0.40 vs. 0.56% for CLA and 0.69 vs. 0.97% of FA for TVA). 116 Inclusion of 4% algae in the diet increased CLA and TVA contents in milk fat by 567% and 425%, respectively, of amounts when no algae was fed. 124 Plant Oil Seeds Plus Marine Oils Researchers have also attempted to enhance CLA in milk fat by feeding combinations of fish and soybean oils or meals, but results have varied. In some studies, fish oil/fish meal was more effective at enhancing the CLA content of milk than adding similar amounts of soybean oil or combinations of fish oil and soybean oil through extruded soybeans or soybean meal. 114,115,118,123 Contrary to the results of these studies, however, are those obtained by AbuGhazaleh et al. 116 In their study, treatment diets were control, 0.5% fish oil through fish meal, 2.5% soybean oil from extruded soybeans, and 0.5% fish oil from fish meal and 2% soybean oil from extruded soybeans. Total milk fat CLA contents were 0.40, 0.56, 0.91, and 1.59% of total FA for the four treatments, respectively. It is worth stating here that a component of fish oil may inhibit the growth of bacteria or production of bacterial enzymes responsible for the reduction of TVA to C 18:0, creating conditions that are more favorable to the later tissue production of CLA from TVA. Therefore, C 18:2 and C 18:3 FA that were provided by the soybeans in the diet indirectly increased CLA synthesis. To further investigate the effect of feeding fish oil along with other fat sources on CLA content of milk, four different fat sources were fed, so that each diet contained 2.0% fat from one of the four fat sources and 1.0% fat from fish oil. 125 The four fat sources were a fat source high in C 18:0, high-c 18:1 sunflower seeds, high-c 18:2 sunflower seeds, and high-c 18:3 flaxseeds. Sunflower and flaxseed shells were cracked by rollers. Milk fat CLA and TVA contents were 0.81, 1.21, 1.94, and 1.21% and 1.64, 2.49, 3.74, and 2.41% of FA for the four treatments, respectively. The highest CLA and TVA contents were observed in milk fat from cows fed fish oil plus high-c 18:2 sunflower seeds. The small increase observed in the flaxseed treatment was probably

8 470 T. R. DHIMAN ET AL. because flaxseeds were small and remained intact during processing, thereby making the fat less available for CLA production in the rumen. Fish or marine oils are usually rich in long chain PUFA (Table 1). The n-3 PUFA content in menhaden fish oil consists largely of C 20:5 (14%), C 22:6 (10%), and C 18:3 (1.0%) 126 as a proportion of total fatty acids. The C 20:5 and C 22:6 fatty acids present in fish oil and fish meal are known to be resistant to ruminal biohydrogenation in vitro; therefore, they are unlikely to be converted directly into CLA. 127 However, as discussed previously, feeding fish oil to dairy cows increases the CLA and TVA contents in milk fat. Feeding fish oil has also been shown to increase the proportion of TVA in rumen digesta, 128 probably through inhibition of the reduction of TVA to C 18:0 in the rumen (Figure 2). The ruminal biohydrogenation of the PUFA in fish oil is not understood well. In the rumen, the inhibitory effect observed when feeding fish oil could be due to the inhibition of the growth of bacteria or production of bacterial enzymes responsible for the reduction of TVA to C 18:0. The origin of CLA in milk fat from cows fed fish oil is very possibly through the desaturation of TVA in the mammary gland by the 9 -desaturase enzyme. The relationship between milk fat CLA and TVA is linear across a wide variety of feeding conditions. 120 However, it has also been shown that the CLA:TVA ratio is lower in milk fat from cows fed fish oil compared to milk fat from cows that are fed plant oils. 120 These findings suggest that a large amount of TVA is produced in the rumen of cows fed fish oil and may exceed the desaturation capacity of 9 -desaturase in the mammary gland, 71 resulting in high levels of TVA in milk fat. It is also possible that certain fatty acids (especially PUFA) from fish oil may inhibit 9 -desaturase activity in the mammary gland. Further research is needed to understand the mechanisms involved in the production of TVA and CLA in the rumen and mammary gland of cows fed fish oil. Fish Oil and Milk Fat Depression As mentioned previously, the reduction of milk fat percentage is a common problem when feeding fish oil to lactating dairy cows and can influence the total CLA yield. Milk fat content was reduced by 20 to 25% when cows were fed diets containing 1.6 to 2.0% fish oil or 4% algae on a DM basis. 114,118,121,122,124 Chilliard and Doreau 129 observed a larger decrease (35%) in milk fat content when mid-lactation cows were fed fish oil at 1.6% of diet DM compared to the milk fat in control cows. However, the increase in the CLA content of milk fat due to feeding fish oil is still larger than the observed decrease in milk fat content. Dairy producers would have to analyze their particular situation to determine the cost and benefits of feeding fish oil. The mechanism by which fish oil decreases milk fat is not clearly understood. However, various explanations have been proposed. Feeding fish oil to dairy cows results in the production of t-c 18:1 FA in the rumen. There is a positive correlation between the concentration of t-c 18:1 FA and milk fat depression. 73,128 It is possible that one or more trans isomers of C 18:1 are responsible for milk fat depression. Specifically, the t10 C 18:1 isomer has been shown to decrease milk fat content in cows fed low fiber diets. 73 The other explanation is that feeding fish oil increases the t10, c12 isomer of CLA. 118 The t10, c12 CLA isomer is also responsible for decreasing milk fat content by reducing de novo synthesis of milk fat in the mammary gland. 102 Animal Fat Supplementing dairy cattle diets with animal fat has the potential to increase the CLA content in milk. Animal fat may sometimes be a source of TVA and CLA that could ultimately become sources of CLA in the mammary gland. In general, fat of ruminant origin is high in C 18:1 and C 16:0 FA (Table 1). Feeding diets containing 3 to 6% tallow to dairy cows increased milk CLA from 0.22% up to 1.10% of fat. 108,130,131 Pantoja et al. 132 fed 5% tallow on a DM basis to dairy cows and observed an increase in TVA from 0.89 to 1.53% of milk fat. However, the authors did not report milk CLA contents in this study. Feeding supplemental fat at 3% of dietary DM through tallow and fish oil in a 33:67 ratio to dairy cows increased the CLA content in milk fat to 2.24% compared to tallow alone (1.1% of fat). 130 Milk fat content was not affected by diet. The CLA content in tallow ranges from 0.29 to1.25% of fat, depending on the animal s diet. 1,133 However, the abomasal infusion of 150 g/d of tallow did not yield higher CLA content in milk compared to a control (0.61 vs. 0.59% of total FA). 110 This is probably explained by the fact that the rumen is the major site of CLA precursor formation. Average increases in milk CLA content from feeding tallow are small when compared to the increases that are seen when grazing cows on pasture. Other Factors Cow Management Systems Dairy cow management systems also influence the CLA content of milk. Jahreis et al. 88 collected milk samples over a period of one year from three farms with different management systems: 1) conventional farming with indoor feeding using preserved forages; 2) conventional farming with grazing during the summer season; 3) ecological farming with no use of chemical fertilizers to produce forages and grazing during the summer season. The CLA content was 0.34, 0.61, and 0.80% of fat in milk from cows fed indoors, grazed during summer, and cows grazed in ecological farming conditions, respectively. Reasons for these results could be due, in part, to differences in vegetation or forage quality among the three systems. Therefore, most of the time, differences in CLA content of milk from cows under different management systems are actually due to the differences in feedstuffs produced under different management styles. An abrupt change in diet of dairy cows from indoor winterfeeding (grass silage, hay, and beets) to pasture grazing sharply increased the level of conjugated dienes in milk fat. 134 Depending on the season, CLA content in milk varied from 0.6 to 1.2% of milk fat, with content being higher in spring and summer than

9 in winter These data suggest that the availability of fresh forages in spring and summer increases CLA content in milk fat compared to mature forages in late summer or conserved forages in winter. There is no difference in conjugated diene content of milk fat between morning and evening milkings. 138 The effects of restricting feed intake on the CLA content of milk have not been clearly identified. When cows were fed a diet in restricted or unrestricted amounts (16.3 vs kg of feed DM/cow per day), milk fat from cows with restricted intake contained twice as much CLA (1.13%) as milk fat from cows with unrestricted intake (0.66% of FA). 72 However, milk yield was slightly lower for restricted cattle. Timmen and Patton 139 restricted feed intake in dairy cows at a more severe level and also observed twice as much CLA (0.46 vs. 0.26% CLA) in milk fat of feed restricted cows compared to milk fat from control cows. It is prudent to state that neither of these studies examined the effects of feed restriction on body condition score. In addition, forage to concentrate ratios were different between treatments, and the authors noted that this probably contributed to their results. In another study, decreasing the grass allowance from 24 to 16 kg/cow per d resulted in a decrease in CLA content of milk from 0.55 to 0.39% of fat. 111 The decrease in the CLA content of milk in this study can be explained by a decrease in the amount of fresh grass intake rather than feed restriction. Restricting feed intake can influence the ruminal biohydrogenation of lipids by effecting changes in ruminal fermentation characteristics and metabolism. Also, it is likely that restricting feed intake would increase the mobilization of body fat in order to meet the animal s energy demand. Mobilized body fat would increase the supply of FA, such as CLA and TVA, to the mammary gland, and therefore increase the CLA content of milk. The magnitude of increase in CLA content will depend on the degree of feed restriction, components of the diet that are restricted, and body fat mobilization. Elevation above sea level was investigated as a possible factor influencing the CLA content of milk. 140 Milk samples were taken from several dairies during the grazing season in the lowlands ( m elevation), mountains ( m), and the highlands ( m) of Switzerland. Milk fat CLA contents were 0.85, 1.58, and 2.34% for the three geographical locations, respectively. Variation in CLA content could be due to differences in plant species and plant fatty acid composition among the three locations. However, there could also be some unexplained differences in fatty acid synthesis or activity of the desaturase enzyme in cows grazing at the three elevations. Cow Breed, Age, and Individual Variation Recent studies suggest that dairy cow breed can also influence the CLA content of milk. Montbeliard cows displayed a tendency to have higher CLA in milk fat (1.85%) compared to Holstein-Friesian (1.66%) or Normande cows (1.64%) grazing on pasture. 141 Holstein-Friesian cows had higher CLA content in milk compared to Jerseys fed diets containing conserved forages and grains 131,142,143 Conjugated linoleic acid content was LINOLEIC ACID CONTENT IN MILK AND MEAT 471 also higher in milk fat from Holstein-Friesians (0.57%) than for Jersey cows (0.46%) when grazed on pasture. 90 Brown Swiss cows had higher CLA content in milk fat than Holstein-Friesian when fed similar diets. 118,142,143 However, Kelsey et al. 144 found that Holstein-Friesian and Brown Swiss cows fed diets containing conserved forages and grain produced milk fat with similar CLA (0.44% and 0.41% of fat, respectively). Ayrshire cows had higher CLA content in milk fat (0.68% of fat) compared to Guernsey and Jersey cows (0.34% of fat) when fed conserved forages at 34% and a grain mixture at 66% of dietary DM. 143 The average difference in CLA content of milk fat among Brown Swiss, Holstein-Friesian, and Jersey breeds is 15 to 20% when fed similar diets. Brown Swiss cows have inherently higher CLA in milk fat, followed by the Holstein-Friesian and Jersey breeds. The data on other breeds are too limited to form any firm conclusions. Preliminary work by Medrano et al. 145 shows that there are differences between Brown Swiss, Holstein-Friesian, and Jersey breeds with respect to the activity of the mammary enzyme stearoyl Co-A desaturase. This information is important, because stearoyl Co-A desaturase oxidizes C 16:0 and C 18:0 to C 16:1 and C 18:1 and is involved in CLA production. Beaulieu and Palmquist 146 and White et al. 90 reported that Jersey cows produced 15 and 13% less C 18:1 than Holstein cows fed similar diets, respectively, confirming the observation of Medrano et al., 145 that mammary desaturase activity differs among breeds. Further understanding of the activity of the desaturase enzyme may offer an explanation as to why there are breed differences in milk fatty acid composition, including CLA. Existing data on the relationship between the age of the cow (lactation number) and CLA content in milk fat show variable results. When cows were fed grass-based diets, cows in the fifth lactation or higher had more CLA content in milk (0.59% of fat; p <.06) than cows in lactations 2 to 4 (0.41% of fat). 111 However, when cows were fed diets containing full-fat rapeseed, there was no indication of a relationship between lactation number and CLA content in milk fat. 111 In another study, older cows (>7 lactations) had higher CLA in milk than younger cows (1 3 lactations). 147 Age differences in milk fat CLA content could be due to differences in desaturase enzyme activities and/or fatty acid metabolism and synthesis between older and younger cattle. Further research is needed to understand the mechanisms involved in differences in CLA production with age of the cow. The CLA content in milk varies from cow to cow, even when the same diet is fed. Jiang et al. 72 and Stanton et al. 111 found substantial variation in the CLA content of milk (0.15% to 1.77% of fat) among individual cows fed the same diet. Kelly et al. 89,98 observed a three-fold variation in CLA content of milk among individual cows fed the same diet at a similar stage of lactation and producing milk with similar fat content. These differences could be due simply to differences in desaturase enzyme activities in the mammary gland, age of animals, disease conditions, differences in ruminal metabolism, or other unknown factors.

10 472 T. R. DHIMAN ET AL. Ionophores and Synthetic CLA Supplements Ionophores have been tested in dairy cows as a potential means of enhancing the CLA content of milk, but results have varied. Feeding ionophores inhibits the growth of gram-positive bacteria, which are involved in ruminal biohydrogenation. Fellner et al. 148 reported that the use of ionophores (nigericin, monensin, and tetronasin) increased the proportion of the c9, t11 isomer of CLA by 200% in fermenter culture in the presence of C 18:2 FA by reducing the complete biohydrogenation of C 18:2, and subsequently resulting in decreased C 18:0 and increased TVA production. Sauer et al. 149 fed 380 mg monensin/cow per d and observed that concentrations of C 18:2 conjugated dienes and TVA in milk fat were increased by 63 and 198%, respectively. However, Dhiman et al. 86 and Chouinard et al. 92 reported little effect on the CLA content of milk fat from cows receiving 250 g or 20 mg of monensin/cow per day, respectively. The variability in results due to the addition of ionophores could be related to dose used or a decrease or a modification in the population of bacteria responsible for biohydrogenation. It is also possible that there is a lack of substrate as suggested by Fellner et al. 148 and Bauman et al. 47 Further studies are needed to elucidate the influence of ionophores on specific bacteria in the rumen, especially bacterial species responsible for lipid biohydrogenation. Synthetic CLA isomers were infused post-ruminally or fed in a ruminally protected form to avoid ruminal biohydrogenation and enhance CLA in milk fat. The administration or feeding of CLA supplements to dairy cows caused a dramatic reduction in the fat content and total yield of milk fat, but resulted in a small increase in milk CLA content. 102,110, Post-ruminal infusion of 150 g/cow per d CLA-60 (60% CLA supplement) to cows for 5dresulted in a linear decrease in milk yield and a 53% reduction in milk fat content, but increased CLA from 0.54% in control cows to 1.91% CLA in milk fat from treated cows. 151 Similar responses in milk fat and CLA content from post-ruminal infusion of CLA supplements were confirmed by others. 152,156 Interestingly, the apparent transfer efficiencies from the CLA infused to CLA in milk were 22 and 10% for c9, t11 and t10, c12 isomers of CLA, respectively, suggesting that infused CLA is extensively metabolized in the body. 151 In another study, the transfer efficiencies of infused CLA for individual isomers were 25.2% for c8, t10; 33.5% for c9, t11; 21.0% for c10, t12; and 28.4% for c11, t13 CLA isomers. 152 Feeding ruminally protected CLA supplement at levels of 30.4 to 100 g/cow per d resulted in a reduction of milk fat content by 27% and increased the total CLA (c9, t11 plus t10, c12 isomers) content of milk fat. 153,154,155,157 The highest transfer efficiencies for c9, t11 and t10, c12 isomers from supplement to milk were 11 and 4%, respectively. 153,154 The low transfer efficiencies seen when feeding ruminally protected CLA supplements compared to abomasally infused CLA are probably due to the incomplete protection of CLA supplements from ruminal biohydrogenation. For lactating goats fed ruminally protected CLA (80 g/animal per d), the CLA content of milk fat was enhanced from 0.6% in control to 4.0% of milk fat in treated animals with a transfer efficiency into milk fat of 39 and 26%. The c9, t11 and t10, c12 CLA isomers, respectively. 158 The reasons for greater transfer efficiencies in goats compared to cows are not clear and merit further research. It is apparent from the existing literature that the infusion or feeding of partially ruminally-protected CLA supplements increases the CLA content of milk fat, but reduces the total milk fat content. Therefore, any increase in the CLA content of milk due to the use of synthetic CLA supplements should be evaluated based on the total CLA yield in milk rather than on content. The mechanisms by which CLA supplements reduce milk fat have been studied. Baumgard et al. 159 abomasally infused relatively pure t10, c12 CLA isomer into dairy cows (at 0.05% of dietary DM) for 4 d. Milk fat content and yield were reduced by 42 and 44%, respectively. In contrast, the infusion of a similar amount of c9, t11 CLA had no negative effect on milk fat content or yield. Similar observations were reported by Loor and Herbein. 102 Data from several studies using relatively pure isomers of CLA suggest that isomers of CLA or their metabolites containing a double bond at the 10th position may have inhibitory effects on milk fat synthesis. 78,151,159,160 Thus, the t10, c12 isomer of CLA present in CLA supplements is most likely responsible for the reduction in milk fat. A summary of factors affecting the CLA content of milk fat is presented in Table 2. CLA CONTENT IN MEAT Although there is a vast amount of literature available about the CLA content of milk, the number of research trials focusing on factors affecting the CLA content of meat is limited. Dietary Factors Affecting CLA in Meat Pastures and Conserved Forages As is the case with dairy cattle, grazing animals on pasture, feeding fresh forages, or increasing the amount of forage in the diet will elevate the percentage of CLA as a proportion of total FA in meat from ruminants. Grazing beef steers on pasture or increasing the amount of silage in the diet increased the c9, t11 CLA content in fat by 29 to 45% compared to control. 161,162 The increase in beef CLA content varies with the quality and quantity of forage in the animal s diet. Beef from steers raised on green pasture had 200 to 500% more c9, t11 CLA as a proportion of fat compared to steers fed an 87% corn grain-based feedlot diet. 163,164 Rule et al. 165 observed that the percentage of c9, t11 isomer of CLA was higher in intramuscular fat of range cattle compared with that of steers fed a high-grain diet under feedlot conditions. The increase in c9, t11 CLA content in beef is not as dramatic as the increase seen in milk from cows grazed on pasture. This difference is probably due to differences in CLA production in the rumen or endogenous synthesis of CLA in intramuscular fat of beef cattle fed high-forage diets.