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ISSN: 1040-8398
DOI: 10.1080/10408390591034463
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-84322-4815, 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 Hilditch2 showed that feed-ing 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 (C18:3),
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, 84322-4810.
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-84322-4815, USA. E-mail: trdhiman@cc.usu.edu
nants. The first evidence of ruminal biohydrogenation of dietary lipids was provided by Reiser4, Hartman et al.,5and Shortland
et al.6It was also established that the process of
biohydrogena-tion in the rumen was incomplete, and that unsaturated fatty acids were saturated by ruminal microoganisms.5The presence of conjugated unsaturated fatty acids in milk was first observed by Booth et al.,7 who reported that milk fat from cows graz-ing pasture in the summer showed an increased absorption in the ultraviolet region (230 nm) as compared to milk fat pro-duced 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, Parodi8isolated cis-9, trans-11 C
18:2(c9, t11
CLA) from milk fat and suggested that fatty acids with con-jugated 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 in-hibited 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 under-stand 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
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properties in various studies in animal models.10−22The mecha-nisms 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.23In several human
can-cer studies, an inverse association was found between the level of CLA in the diet and the risk of developing cancer in breast adipose tissue.24−27
Studies conducted with mice, chickens, and pigs have sug-gested a possible role of CLA (mainly the trans-10, cis-12 iso-mer) in decreasing body fat and increasing lean body mass.28−33
A human-related study has suggested that CLA increases body mass without increasing body fat.34Several studies indicate that
CLA may enhance immune function.35−40Conjugated linoleic acid has also been found to have antidiabetic and antiatheroscle-rotic properties in animal models.41−45
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.47published an excellent article de-scribing the biosynthesis of CLA in ruminants. Other articles have focused on the potential health benefits of CLA.48,49The 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 in-fluence the CLA content of milk and meat, as well as processed products, thereby making them even better food sources for hu-man consumption.
CLA ISOMERS
Conjugated linoleic acid is a collective term for a series of conjugated dienoic positional and geometrical isomers of linoleic acid (C18:2). Conjugated linoleic acid isomers are found
naturally in foods, especially those of ruminant origin.1 In ru-minants, CLA is synthesized by ruminal bacteria using C18:2or
C18:3as the precursor.50 Conjugated linoleic acid isomers can
also be synthesized in the laboratory from C18:2or from sources
high in C18:2, such as sunflower, safflower, soybean, or corn
oils, by a reaction involving alkaline water isomerization51 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.8,53−55In recent years, biological activities have been proposed for other CLA isomers, especially
trans-10, cis-12 C18:229,30(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 C18:2are shown in Figure 1.
A total of 17 natural CLA isomers have been found in milk, dairy products, beef, human milk, and human adi-pose tissue using silver ion-high performance liquid chro-matography and gas chrochro-matography-electron ionization mass spectrometry.1,52,56−61Identified 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.52identified 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 iso-mer (72%), followed by the t7, c9 isoiso-mer (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 Fritsche63 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 bio-hydrogenation of C18:2and C18:3or from endogenous synthesis
in tissues as shown in Figure 2. Ruminally, CLA is produced as an intermediate product during the biohydrogenation of di-etary C18:2or C18:3to stearic acid (C18:0). Endogenously, CLA is
synthesized from t11, C18:1vaccenic acid (TVA), another
inter-mediate of ruminal biohydrogenation, via9-desaturase.47The 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
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Figure 1 Abbreviated chemical structures of ordinary C18:2(linoleic acid) (A) and two major conjugated linoleic acids: c9, t11 isomer (B) and t10, c12 isomer
(C).
ranges from 3–7% on a dietary dry matter (DM) basis. The fatty acid profiles of some common ruminant feeds are pre-sented in Table 1. Most ruminant feeds of vegetable origin con-tain C18:2and/or C18:3as the predominant fatty acids. Feeds of
animal origin, such as tallow and fish products, are likely not to
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
be as rich in these fatty acids. Among feeds, pasture diets fed to ruminants are rich in C18:3, representing 48 to 56% of total
fatty acids (FA). Corn or grass silages are rich in C18:2 (41%
of FA) or C18:3(46% of FA), respectively. Alfalfa hay contains
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Table 1 Fatty acid profile of common ruminant feeds
Feed∗ C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 Others∗∗
— Fatty acid, % of total reported fatty acids —-Pasture Grass208 0.5 19.2 0.2 1.6 2.2 20.4 55.9 0.0 Clover208 0.5 22.9 0.3 3.4 3.6 21.1 48.2 0.0 Grass+ legume86 1.5 20.0 1.2 2.6 4.2 18.9 51.6 0.0 Silage Grass163 5.4 24.0 0.6 2.9 6.3 14.5 46.2 0.0 Corn123 1.1 15.2 0.5 3.5 18.9 40.9 6.1 13.8 Hay alfalfa123 1.2 22.9 0.4 4.0 4.9 18.1 23.5 25.0 Concentrates Barley209 0.0 27.6 0.9 1.5 20.5 43.3 4.3 1.9 Corn209 0.0 16.3 0.0 2.6 30.9 47.8 2.3 0.0 Oats209 0.0 22.1 1.0 1.3 38.1 34.9 2.1 0.5 Wheat209 0.0 20.0 0.7 1.3 17.5 55.8 4.5 0.2 Byproducts Gluten meal209 0.0 17.2 0.9 0.8 26.7 53.0 1.4 0.0 Distillers’ grains209 0.0 15.6 0.0 2.7 24.2 54.5 1.8 1.2 Plant seeds/oils Soybean108 0.0 11.0 0.0 3.8 23.3 54.5 5.9 1.5 Extruded 0.0 14.5 0.0 3.8 19.5 53.2 9.1 0.0 soybean99 Extruded 0.0 23.4 0.5 2.2 16.5 57.4 0.0 0.0 cottonseed99 Sunflower98 0.0 4.0 0.0 5.4 21.2 69.4 0.0 0.0 Peanut98 0.0 12.3 0.0 3.2 51.5 30.2 0.0 2.8 Linseed98 0.0 6.5 0.0 4.0 22.7 15.4 51.4 0.0 Fish oil126 8.0 22.0 11.0 3.0 21.0 2.0 1.0 32.0*** Animal tallow210 3.2 24.8 5.3 14.5 45.9 5.9 0.3 0.0
∗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 C20:5(14%), C22:6(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 C18:2, accounting for 53 to
69% of total FA (Table1). However, peanut oil is rich in C18:1
(51%) and linseed oil contains an abundance of C18:3(51% of
total FA). Fish oil contains relatively low amounts of C18:2and
C18:3, but is very rich in long chain polyunsaturated fatty acids
(PUFA). Animal fat has a high proportion of C18:1(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.68In the second
pro-cess, the unsaturated free FA are rapidly hydrogenated by mi-croorganisms in the rumen to produce more highly saturated end products.
The c9, t11 isomer of CLA is the first intermediate prod-uct in the biohydrogenation of C18:2 by the enzyme linoleate
isomerase (Figure 2), which is produced by the microorganism
Butyrivibrio fibrisolvens50 and other bacterial species. Part of the c9, t11 CLA is rapidly reduced to TVA or C18:0,69,70
becom-ing available for absorption in the small intestine. Similar to the biohydrogenation of C18:2, the FAα and γ –C18:3, which are the
predominant unsaturated FA in forages, also undergo
isomer-ization and a series of reductions, ending with the formation of C18:0 in the case of complete biohydrogenation.71 The c9, t11
CLA and TVA often escaping complete ruminal biohydrogena-tion 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 C18:2to t-C18:1
and related isomers, but only a few have the ability to reduce C18:2 and C18:1 completely to C18:0.74 Interestingly, no single
species of rumen bacteria catalyzes the complete biohydrogena-tion 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 the9-desaturase
enzyme.64,65,79
Several studies have been performed to confirm that the en-dogenous synthesis of CLA occurs in the mammary gland by
9-desaturase. Trans-vaccenic acid (12.5 g/d) was infused
abo-masally 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 pro-duction was increased by 17% in milk fat.65In addition, specific
inhibitors of9-desaturase, such as sterculic oil [sterculic acid
(C19:1) plus malvalic acid (C18:1)] or sterculic acid only, were
in-fused 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 the9-desaturase
enzyme is responsible for the introduction of a cis-double bond between carbons 9 and 10 of the FA. The actual estimated en-dogenous synthesis of c9, t11 CLA in milk fat was 64,6478,65or
80%,81of 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 distribu-tion of9-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.82−84In lactating ruminants, the highest activity of
9-desaturase is found in the mammary tissue.85There is very
little research exploring the factors that influence and regulate
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is also needed to gain a better understanding of the influence of the level of9-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.87−90
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 48 to 56% of the total FA in fresh forages consists of C18:391
(Table 1). Fresh grass supplies C18:3FA as a substrate for
ru-minal biohydrogenation. However, the abundant supply of C18:3
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 produc-tion. Ruminal pH is generally relatively high in cows grazing pasture compared to cows fed a combination of conserved for-age 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.86Similarly, 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 ad-dition of grain to dairy diets decreases ruminal pH. A decrease in pH will change the microbial population and affect ruminal fermentation.77It has been suggested that the main ruminal
bio-hydrogenating bacteria are cellulolytic.93,94Reduction in rumi-nal pH decreases the population of cellulolytic bacteria and other microbes responsible for lipid biohydrogenation and the produc-tion of CLA and TVA.72
Table 2 Factors affecting conjugated linoleic acid (CLA) content of milk Total CLA Factors∗ (% of fat) Diets Forage Freshness: Pasture86−90 0.59–2.21 Silage86,88 0.34–0.86 Hay86 0.79 Maturity: Heading92 1.14 Second cutting92 0.81 Flowering92 0.48
Plant seed oils
Soybean oils (3–4%)101,105 0.71–2.13 Linseed oil (4.4–5.3%)98,105 1.67–1.70 Peanut oil (5.3%)98 1.33 Sunflower oil (5.3%)98 2.44 (2.5%)102 0.72 Canola oil (3–3.3%)101,109 0.51–1.10 Canolamide (3.3%)109 0.70 Safflower oil (2.5%)100,102 0.61–1.00 Infusion (150 g/d)110 0.58 Ca-treated oils (4%):
Canola, soybean, linseed108 1.32–2.25
Intact oil seed
Raw soybean (17.5%)108 0.31
Full fat rapeseed (0.83–1.65 kg)111,112 0.52–2.49
Full fat extruded soybean
(1.65 kg)112 2.23
(10.6–17.5%)99,108,113−118 0.73–1.24
Full fat extruded cottonseed (12%)99 0.60
Whole ground flax, solin, or canola87,119 1.16–1.49 Fish oils/meals Fish oils (1–3%)114,115,118,121,122 1.00–2.53 (200–400 ml/d)108 1.65–1.74 Fish meals (3–5.8%)86,116,123 0.56–0.86 Algae (4%)124 2.47 Animal Fat Tallow (3–6%)108,130,131 0.47–1.1 Infusion (150 g/d)110 0.61
Combinations of oilseeds and fish oil/meal Extruded soybean and fish oil
5.3% soybean and 1.0% fish oil115,118,125 0.82–2.17
10.6% soybean and 1.0% fish meal116 1.59
Sunflower/flaxseeds and fish oil
4–5% and 1.0%, respectively125 1.21–1.94 Management system Seasonal effects Spring135,137 0.60–1.00 Summer134,135,137 1.20–1.70 Winter134,135,137 0.60–0.80 Fall135,137 0.90–1.20 Elevation 600–650 m140 0.85 900–1210 m140 1.58 1275–2120 m140 2.34 Restricted Feeding Unrestricted72,111,139 0.26–0.66 Restricted72,111,139 0.39–1.13 Breed Holstein Pasture90,141,143 0.72–1.66
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Table 2 Factors affecting conjugated linoleic acid (CLA) content of milk (Continued) Total CLA Factors∗ (% of fat) TMR90,118,131,142,144 0.41-0.93 Brown Swiss Pasture143 1.22 TMR118,142,144 0.41–0.71 Jersey Pasture90,143 0.59–0.77 TMR90,131,142 0.45–0.72 Ayrshire (Pasture)143 0.57 Guernsey (Pasture)143 0.36 Montebeliardes (Pasture)141 1.85 Normandes (Pasture)141 1.64
Age or lactation number
Aged cow (>4 lactations)111 0.59
Young cow (2–4 lactations)111 0.41
Ionophores
Monensin (20–380 mg or 250 g)86,92,149 0.42–1.30
Synthetic CLA supplements Infusing (post-ruminally) CLA-60 (150 g/d)151 1.91 CLA-46 (28.8 g/d)152 1.02 CLA-47 (248.5 g/d)152 1.52 CLA-88(16.3 g/d)152 0.95 Cis9, trans 11 (15 g/d)102 0.87 Trans10, cis 12 (15 g/d)102 0.85 Mixture 20–40 g/d (pasture-fed)156 2.56–2.98 150 g/d110 2.62 Feeding CLA-60 (100 g/d)153,154 0.59–0.77 CLA mix (30.4 g/d)155 0.55
∗Numerical superscripts next to factors correspond to reference numbers cited
in the reference section.
Additionally, the endogenous production of CLA in the mam-mary glands of pasture-fed cows cannot be excluded. The9
-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
18:3content 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 C18:3
and total FA in grass, whereas harvesting forage as silage, when carried out properly, does not.95 The content of C18:3FA may
decrease when forage is wilted before ensiling, or if there is undesirable fermentation during ensiling.96,97 The amount of C18:3FA 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.98−102These oils are rich in C18:2and C18:3FA. Studies have found that high
levels of C18:2and C18:3(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 C18:2 inhibits the
final reduction of TVA, thus increasing its accumulation in the rumen,73and subsequent availability to the animal.
Feeding diets containing soybean oil (4%) resulted in approx-imately a four-fold increase in CLA content of milk fat (2.08%) over the control (0.50% of milk fat).105Supplementing 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
Feed-ing 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 C18:2are more
effective at increasing CLA in milk fat as compared to oils rich in C18:3or C18:1. Dhiman et al.105reported 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
Herbein101 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 C18:2produced more CLA in milk fat than cows fed
similar oils that were high in C18:1100,102The reason why C18:2
produces more CLA than C18:1is 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 C18:1is primarily
hydrogenated to C18:0or isomerized to various t C18:1isomers
(mainly t-4 C18:1to t-10 C18:1),107rather 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.108The five-fold
increase of CLA in this study suggests that the calcium salts of FA were not protected from ruminal biohydrogenation. Loor et al.109investigated the effects of feeding protected or
unpro-tected canola oil on milk fatty acid composition. Cows were fed a control diet; a diet containing canola oil at 3.3% of di-etary DM, a diet containing canolamide (made by a reaction of canola oil and ethanolamine to protect oil from ruminal biohy-drogenation) 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,
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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 suc-cessful at increasing the CLA content of milk fat.110 This is strong evidence that ruminal biohydrogenation of C18:2plays a
major part in the synthesis of CLA that is eventually incorpo-rated 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.87,99,108,111−118
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.113Feeding 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.108Extruding 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 C18:3
(102 g/d) from green grass had higher milk CLA content (2.21% of fat)86than cows fed diets containing conserved forages and
grain supplemented with C18:3(575 g/day) through linseed oil
(1.67% CLA in milk fat).98However, it should be acknowledged
that factors other than oil supply from pasture grass are also re-sponsible 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 2–2.5 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.
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.121Feeding 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.86,105,123In 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).116Inclusion 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 re-sults have varied. In some studies, fish oil/fish meal was more ef-fective 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 ob-tained by AbuGhazaleh et al.116In 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 con-tents 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 compo-nent of fish oil may inhibit the growth of bacteria or production of bacterial enzymes responsible for the reduction of TVA to C18:0, creating conditions that are more favorable to the later
tissue production of CLA from TVA. Therefore, C18:2and C18:3
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.125The four
fat sources were a fat source high in C18:0, high-C18:1sunflower
seeds, high-C18:2sunflower seeds, and high-C18:3flaxseeds.
Sun-flower 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, respec-tively. The highest CLA and TVA contents were observed in milk fat from cows fed fish oil plus high-C18:2sunflower seeds. The
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because flaxseeds were small and remained intact during pro-cessing, thereby making the fat less available for CLA produc-tion 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 con-sists largely of C20:5(14%), C22:6(10%), and C18:3(1.0%)126as
a proportion of total fatty acids. The C20:5and C22:6fatty acids
present in fish oil and fish meal are known to be resistant to ru-minal biohydrogenation in vitro; therefore, they are unlikely to be converted directly into CLA.127However, as discussed
pre-viously, 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,128probably
through inhibition of the reduction of TVA to C18:0in 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 C18:0.
The origin of CLA in milk fat from cows fed fish oil is very possibly through the desaturation of TVA in the mam-mary gland by the9-desaturase enzyme. The relationship be-tween milk fat CLA and TVA is linear across a wide variety of feeding conditions.120However, 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.120These
findings suggest that a large amount of TVA is produced in the rumen of cows fed fish oil and may exceed the desaturation ca-pacity of9-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 inhibit9-desaturase
activity in the mammary gland. Further research is needed to un-derstand 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 Doreau129observed 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 pro-duction of t-C18:1 FA in the rumen. There is a positive
cor-relation between the concentration of t-C18:1FA and milk fat
depression.73,128It is possible that one or more trans isomers of C18:1are responsible for milk fat depression. Specifically, the
t10 C18:1isomer has been shown to decrease milk fat content in
cows fed low fiber diets.73 The other explanation is that
feed-ing fish oil increases the t10, c12 isomer of CLA.118The t10,
c12 CLA isomer is also responsible for decreasing milk fat
con-tent by reducing de novo synthesis of milk fat in the mammary gland.102
Animal Fat
Supplementing dairy cattle diets with animal fat has the po-tential 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 C18:1and C16:0FA (Table 1).
Feed-ing diets containFeed-ing 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).130Milk 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.1,133However, 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 con-tent of milk. Jahreis et al.88collected milk samples over a pe-riod of one year from three farms with different management systems: 1) conventional farming with indoor feeding using pre-served 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 winter-feeding (grass silage, hay, and beets) to pasture grazing sharply increased the level of conjugated dienes in milk fat.134 Depend-ing 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
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in winter.134−137These 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. 23.7 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 Patton139
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.111The 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 biohydro-genation 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 mam-mary 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 fac-tor influencing the CLA content of milk.140Milk samples were taken from several dairies during the grazing season in the low-lands (600–650 m elevation), mountains (900–1210 m), and the highlands (1275–2120 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 differ-ences 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 influ-ence 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.141Holstein-Friesian cows had higher CLA content in milk compared to Jerseys fed diets containing conserved for-ages and grains131,142,143 Conjugated linoleic acid content was
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 How-ever, 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.145shows 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, be-cause stearoyl Co-A desaturase oxidizes C16:0and C18:0to C16:1
and C18:1 and is involved in CLA production. Beaulieu and
Palmquist146and White et al.90 reported that Jersey cows pro-duced 15 and 13% less C18:1than 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 of-fer an explanation as to why there are breed difof-ferences 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 num-ber and CLA content in milk fat.111In another study, older cows (>7 lactations) had higher CLA in milk than younger cows (1–3 lactations).147Age 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 in-volved 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.72and Stanton et al.111found
sub-stantial 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 ac-tivities in the mammary gland, age of animals, disease con-ditions, differences in ruminal metabolism, or other unknown factors.
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Ionophores and Synthetic CLA Supplements
Ionophores have been tested in dairy cows as a poten-tial means of enhancing the CLA content of milk, but re-sults have varied. Feeding ionophores inhibits the growth of gram-positive bacteria, which are involved in ruminal biohy-drogenation. Fellner et al.148reported 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 C18:2FA by reducing the complete
biohydro-genation of C18:2, and subsequently resulting in decreased C18:0
and increased TVA production. Sauer et al.149fed 380 mg
mon-ensin/cow per d and observed that concentrations of C18:2
conju-gated 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 pop-ulation of bacteria responsible for biohydrogenation. It is also possible that there is a lack of substrate as suggested by Fellner et al.148and Bauman et al.47Further studies are needed to elucidate the influence of ionophores on specific bacteria in the rumen, es-pecially 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.102,110,150−156
Post-ruminal infusion of 150 g/cow per d CLA-60 (60% CLA supplement) to cows for 5 d resulted 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.152,156Interestingly, the apparent transfer ef-ficiencies from the CLA infused to CLA in milk were 22 and 10% for c9, t11 and t10, c12 isomers of CLA, respectively, suggest-ing 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 con-tent 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 sup-plements compared to abomasally infused CLA are probably due to the incomplete protection of CLA supplements from ru-minal 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 sim-ilar amount of c9, t11 CLA had no negative effect on milk fat content or yield. Similar observations were reported by Loor and Herbein.102Data from several studies using relatively pure
isomers of CLA suggest that isomers of CLA or their metabo-lites 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.161,162The 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.163,164Rule et al.165observed 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.
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Plant Oils and Seeds
Supplementing beef cattle diets with C18:2or C18:3- rich plant
oils has yielded varied results as far as increasing the CLA con-tent of beef. Feeding 4 to 6% of diet DM as soybean oil to beef cattle fed high grain diets either marginally increased or did not increase the c9, t11 CLA content of beef.166−169 There was a small increase in the c9, t11 CLA content of fat from beef mus-cle when steers were fed 3 to 6% sunflower oil compared to beef from cattle fed no oil (0.35 vs. 0.25% CLA in beef fat).170,171
In another study by the same authors, feeding 6% sunflower oil to cattle from Wagyu, Limousin x Wagyu, and Limousin breeds increased CLA (isomer not mentioned) content to 1.25% of fat in beef muscle compared to 0.28% in cattle fed 0% sunflower oil.172Feeding linseed oil at 6% of diet DM increased the c9, t11
CLA content to 0.80% of fat compared to 0.32% of total FA in muscle from control cattle.173The increase seen in CLA content
of beef muscle when including free oils in the animal’s diet is small, with linseed oil or sunflower oil showing more promising effects than soybean oil.
Feeding processed plant oil seeds has also resulted in marginal or no increase in CLA content of beef. Replacing nor-mal corn with high-oil corn or feeding cracked safflower seeds as a source of oil to beef cattle did not increase CLA content in beef muscle.162,174 However, in one study, feeding whole
sun-flower seeds at 5% of diet DM increased the CLA to 0.73% of fat compared to 0.31% CLA in subcutaneous fat in the control group.175Feeding full-fat extruded soybeans at 12.7 and 25.6%
of diet DM to beef cattle fed high grain diets marginally in-creased CLA and TVA contents of beef muscle compared to the control.176
Some researchers have attempted to increase the CLA content of lamb by manipulating the diet. Feeding up to 6% safflower oil, 6% sunflower oil, or adding 5% additional fat through cracked safflower seeds resulted in a two-fold increase in the CLA con-tent of muscle.177−180This is only a small increase when com-pared to the observed increase in CLA content as a proportion of total fatty acids when animals are raised on forages and pasture. However, it is important to realize that total body fat content is decreased when animals are grazed on pasture or fed high forage diets; therefore, actual CLA yields may actually be higher when supplementing oil to grain-based diets compared to grazing an-imals on pasture. Interestingly, the CLA content was increased from 1.0 to 1.6% of fat in muscle when lambs were fed whole linseeds, but there was no increase reported when fish oil was added to the diet.181
Feeding feed sources rich in C18:2 or C18:3 to dairy cows
results in a 3- to 4-fold increase in the c9, t11 CLA content in milk fat,98,105 but only marginal increases in beef fat. It is very possible that the mechanisms and routes of CLA synthe-sis (ruminal and endogenous) are different in the mammary gland and adipose tissue. Additional factors regulating the syn-thesis of CLA in the rumen, muscle, and mammary gland are poorly understood. Further research is also needed to demon-strate synthesis of c9, t11 CLA in the adipose tissue from TVA in ruminants.
Finishing beef cattle are typically fed diets containing 85 to 92% grain,182 whereas a typical diet for a high-producing
dairy cow consists of only 50 to 60% grain. The lower pro-portion of c9, t11 CLA in beef fat compared to milk fat in animals fed diets rich in C18:2or C18:3probably relates to the
effects of the traditional high-grain, low-fiber diets fed to fin-ishing cattle in the United States. It seems likely that the acidic ruminal pH often occurring in finishing beef cattle alters the microbial population involved in lipid biohydrogenation, and therefore influences the ruminal synthesis of CLA isomers. Re-search suggests that high-grain diets resulting in low ruminal pH lead to shifts in the ruminal environment that favor the produc-tion of the t10, c12 CLA isomer and TVA in the rumen,148,183 thereby resulting in higher concentrations of these FA in the beef muscles.167,168
In muscle, the substrate TVA is present; however, it may not be converted to c9, t11 CLA. The t10, c12 isomer of CLA has been shown to inhibit the activity and gene expression of the9
-desaturase enzyme,184,185which would result in a reduction in the endogenous synthesis of c9, t11 CLA. However, this seems an unlikely explanation for no observed increase in c9, t11 CLA in beef fat, because steers finished on pasture or high forage diets show increased percentages of both c9, t11 and t10, c12 isomers of CLA as a proportion of total FA.164
The abundance of mRNA and the enzyme activity of9
-desaturase are also affected by hormone balance, physiolog-ical state, insulin level, and other activating and inhibiting factors.186,187 A decline in insulin resulted in decreased 9
-desaturase gene expression in adipose tissue.188
It seems likely that the greatest potential to increase the total CLA yield of beef would come from supplementing oils, such as soybean oil, linseed oil, or sunflower oil to high-grain diets, although results have not always been positive. Grazing animals on pasture substantially increases CLA as a proportion of total fatty acids, but total fat content in the final product is reduced. Therefore, increase in CLA content of beef should be evalu-ated based on total CLA available in the edible fat rather than concentrations in raw meat.
Animal Breed and Management Strategies
Besides dietary factors, researchers have also studied the in-fluence of beef cattle breed on CLA content in meat. Limited studies suggest that there is little breed effect. The CLA content in beef muscle was similar in European x British crossbreeds and 75% Wagyu cattle fed high-grain, barley-based diets.189 Limousin cattle had only marginally higher CLA content in beef muscle compared to Wagyu and Limousin x Wagyu cattle fed similar diets.172More studies are needed to understand the breed
influence on CLA content of beef.
Recent studies indicate that there may be some influence of the type of management strategy used to raise cattle on the CLA content of beef. Steers were fed a high-grain fin-ishing ration immediately after weaning or a backgrounding diet containing 98% alfalfa silage for 112 d, followed by the
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high-grain finishing diet until slaughter. The CLA content in muscle was modestly increased by feeding the backgrounding diet.190 Apparently, CLA deposited during backgrounding
re-mained in the muscle throughout the remainder of the finishing period. In another study, beef steers receiving backgrounding diets that contained 40% grain and finishing on high grain diets backgrounded and then finished on pasture, or fed all forage di-ets during backgrounding and then finished on pasture had 0.27, 0.86, and 1.53% CLA in beef fat, respectively.164 Despite the fact that the latter two groups were both finished on the same pasture, CLA levels were different. These data suggest that di-etary and management strategies, operating primarily through diet alterations used to raise cattle, can have major influence on the CLA content of beef.
Table 3 summarizes the factors that influence the CLA content of beef. Existing literature indicates that the total CLA content (sum of c9, t11 and t10, c12 isomers) of beef varies from 0.17 to 1.35% of fat. The broad range of CLA content of beef is related to the wide variety of feeds offered, breed differences, and man-agement strategies used to raise cattle. It should be emphasized that data presented in Table 3 report CLA as a proportion of total fat. A large portion of the studies cited did not report total fat content. Therefore, caution should be taken when interpreting these data with respect to total CLA yields.
PROCESSING EFFECTS ON CLA CONTENT Milk Processing
The effects of processing conditions, dairy cultures, and stor-age conditions on CLA content of dairy products have been Table 3 Factors affecting conjugated linoleic acid (CLA) content in beef
Total CLA
Factors∗ (% of fat)
Diet Pasture
Grass only161,163−165 0.48–1.35
Grass and grain161 0.52
Plant seed oils
Linseed oil (6%)173 0.80
Soybean oil (2–5%)166−168 0.29–0.55
Sunflower oil (3–6%)170−172 0.26–1.29
Intact oil seeds
Safflower seed (5%)174 1.10
High-oil corn (74–82%)162 0.38–0.49
Full-fat extruded soybean (13–26%)176 0.69–0.77
Fish oil Fish oil (6%)173 0.57 Breed European x British189 0.17 Wagyu172,189 0.18–0.27 Limousin x Wagyu172 0.28 Limousin172 0.29 Management
High-grain finishing ration190 0.32
Backgrounding followed by high-grain ration190 0.35
∗Numerical superscripts next to factors correspond to reference numbers cited
in the reference section.
studied. Pasteurizing raw milk at 68.3◦C for 30 min did not alter the CLA content of milk.191,192 Processing milk under normal conditions (up to 85◦C for 30 min) into dairy products such as yogurt, ice cream, sour cream, and cheese (Mozzarella, Gouda, and Cheddar) had no influence on the CLA content.99,193,194
The CLA content of dairy products increases when milk is processed at higher temperatures. Clarification of ghee (butter oil) at 110 and 120◦C increased the CLA content to 0.9 and 2.1% of fat, respectively, compared to 0.6% in raw milk.195The CLA content of processed cheese was increased by 14.4% during the normal heating process in the cooker.196In another study, the
processing of Cheddar cheese at 80 to 90◦C under atmospheric conditions increased CLA from 0.40 to 0.51% of fat; however, processing under nitrogen at 70 to 85◦C did not alter the CLA content.197 The CLA content in butter processed at 7 to 11◦C
increased to 0.63% compared to 0.50% of fat in raw milk.193
Using different cheese cultures, processing parameters, and aging (13 mo) had a negligible effect on the CLA contents in Cougar Gold, Cheddar, and Viking cheeses.198Conjugated
linoleic acid as a percent of fat seems to be slightly elevated in very ripe propionic acid fermented cheeses, whereas bacte-rial surface ripened cheeses, such as Muenster and Tilsiter, have intermediate levels of CLA.199Reprocessing cheddar cheese at 80◦C by adding whey protein concentrate or its low molecular weight fraction at 6% by weight increased the CLA content to 0.67 and 0.59% of fat, respectively, compared to 0.50% of fat in the control. This suggests that whey protein concentrate and its low molecular weight fraction may interact with C18:2
radi-cals in processed cheese, resulting in the formation of CLA.197
The CLA content in milk fat increased from 0.6% in raw milk to 1.0% of fat in curd during natural microbial fermentation.195
Storing yogurt, sour cream, butter, or ice cream at 4◦C for 6 wk, and cheese (Mozzarella and Cheddar) for 32 wk did not alter the CLA content.193,200
Existing literature suggests that CLA in milk fat is a sta-ble compound under normal processing and storage conditions; however, processing dairy products at>80◦C may slightly ele-vate the CLA content. There is not enough data available to form conclusions about the effects of different cultures and additives on the CLA content of dairy products.
Limited data suggest that antioxidants or dairy additives may affect CLA contents in processed cheese.201The total CLA
con-tent was increased to 0.43, 0.41, 0.47, and 0.38% of fat by adding ascorbate, cysteine, propyl gallate, or butylated hydroxytoluene, respectively, compared to 0.33% in control with no additive.201
Meat Processing
The influence of cooking temperatures and methods on the CLA content of beef have been studied by a few researchers. They also have been shown to influence the CLA content of beef, as CLA may be formed by thermal oxidation of C18:2or
destroyed by high cooking temperatures and oxidative reactions during subsequent storage. Ha et al.192reported a moderate
