increasing the concentrations of beneﬁcial polyunsaturated ... · increasing the concentrations...

Animal Feed Science and Technology131 (2006) 168–206

Increasing the concentrations of beneficialpolyunsaturated fatty acids in milk produced by

dairy cows in high-forage systems

R.J. Dewhurst a,∗, K.J. Shingfield b, M.R.F. Lee c, N.D. Scollan c

a Agriculture and Life Sciences Division, Lincoln University, Canterbury, New Zealandb MTT Agrifood Research, FIN-31600 Jokioinen, Finland

c Institute of Grassland and Environmental Research, Aberystwyth SY23 3EB, UK

Accepted 6 April 2006

Abstract

There is considerable interest in altering the fatty acid composition of milk with the overall aimof improving the long-term health of consumers. Important targets include reducing the amounts ofmedium-chain saturated fatty acids, enhancing cis-9 18:1 to reduce cardiovascular risk, as well asincreasing concentrations of trans-11 18:1 and cis-9, trans-11 18:2 which have been shown to exertanti-carcinogenic properties in a range of human cell lines and animal models. Most studies haveexamined use of plant or marine oils, vegetable oilseeds or rumen protected or inert lipids in the dietto modify milk fatty acid composition, with much less attention paid to the fatty acid compositionof the basal forage. We review recent progress in this area and identify potential for increasing thelevels of mono- and poly-unsaturated fatty acids (MUFA and PUFA) in milk produced by dairy cowsin high-forage systems. We also review the range of levels of important MUFA and PUFA in milkachieved by feeding the major classes of forage, as well as considering effects of less common foragesto reveal potential new approaches to manipulate rumen fatty acid metabolism. Even though foragescontain relatively low levels of lipid, they are often the major source of fatty acids in ruminant diets.We describe ways in which herbage species, cultivar, conservation method and level of forage in

Abbreviations: C, concentrate; CLA, conjugated linoleic acid; DM, dry matter; DHA, docosahexaenoicacid; EPA, eicosapentaenoic acid; F, forage; HDL, high density lipoprotein; LDL, low density lipoprotein;MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; TMR, totalmixed ration

∗ Corresponding author. Tel.: +64 3 325 3838x8101; fax: +64 3 325 3851.E-mail address: [email protected] (R.J. Dewhurst).

0377-8401/$ – see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.anifeedsci.2006.04.016

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R.J. Dewhurst et al. / Animal Feed Science and Technology 131 (2006) 168–206 169

the diet of dairy cows affect rates and extents of ruminal biohydrogenation of dietary fatty acidsand milk fatty acid composition. Discussion of the potential to increase recovery of forage PUFA inmilk first considers genetic approaches to increase PUFA in forage, including in a range of animalmanagement systems. Losses of fatty acids during forage conservation and storage are described alongwith strategies to reduce such losses. We describe plant traits, such as polyphenol oxidase, tannins andoutflow rates from the rumen, which are associated with reduced rumen biohydrogenation. Similarly,we describe plant factors associated with increased levels of biohydrogenation intermediates in therumen. The second aspect of this review focuses on effects of forage composition on metabolismof supplementary PUFA showing that both the level and type of forage in the diet are importantdeterminants of ruminal lipid metabolism and milk fatty acid responses to plant and marine oils.Whilst increased use of forage in the diet does not produce the large 18:3 n − 3, conjugated linoleicacid or cis-9 18:1 enrichment, or saturated fatty acids depletion in milk, that can be achieved withdietary oil supplementation, beneficial changes can be made without substantial increases in milktrans fatty acids. Trans fatty acids are an important issue for future research, both because of theirgenerally negative effect on human health, as well as because trans-11 18:1 appears to be an exceptionto this generalisation.© 2006 Elsevier B.V. All rights reserved.

Keywords: Forage; Fatty acid; Rumen; Biohydrogenation; CLA

1. Introduction

Fat in milk and dairy products makes an important contribution to consumption ofessential fatty acids and vitamins in the human diet, and plays a critical role in the sen-sory attributes of these foods (Demment and Allen, 2004; Chen et al., 2004; Chilliard andFerlay, 2004). However, there is a consumer perception that ruminant products, such as milk,have a high fat content and they are considered to contribute towards some human diseases.Recent nutritional guidelines for humans in numerous countries emphasise the importanceof maintaining a balanced diet in helping to reduce the incidence of non-communicablediseases such as obesity, type-2 diabetes, cancer and cardiovascular disease (World HealthOrganisation, 2003; Leaf et al., 2003; Jacobs et al., 2004). It is recommended that totalfat, saturated fatty acids (SFA), n − 6 polyunsaturated fatty acids (PUFA), n − 3 PUFA andtrans fatty acids should contribute <0.15–0.30, <0.10, <0.05–0.08, <0.01–0.02 and <0.01of total energy intake, respectively.

In many developed countries, higher consumption of total SFA is associated with milkand dairy products (Hulsof et al., 1999; Valsta et al., 2005). This has contributed to amore negative image for these products. Milk fat typically contains a high proportion ofSFA (0.70–0.75; largely as a consequence of microbial biohydrogenation in the rumen)and monounsaturated fatty acids (MUFA; 0.20–0.25) and small amounts of PUFA (0.05)(Lock and Shingfield, 2004). The medium-chain SFA (i.e., 12:0, 14:0 and 16:0) whichaccount for the majority of SFA in milk fat have been implicated in increasing total and lowdensity lipoprotein (LDL) cholesterol concentrations (Williams, 2000), although Clandininet al. (2000) suggests that 16:0 may not have negative effects if the supply of 18:2 n − 6is adequate. In contrast, stearic acid (18:0) is considered to be neutral in this regard. Oleicacid (18:1 n − 9) is the most prominent MUFA, with the remainder of the MUFA occurring

170 R.J. Dewhurst et al. / Animal Feed Science and Technology 131 (2006) 168–206

mainly as trans isomers of 18:1, although isomers of 16:1 (Destaillats et al., 2000) and20:1 (Cruz-Hernandez et al., 2004) are present, albeit at low concentrations. Linoleic (18:2n − 6) and �-linolenic acid (18:3 n − 3) are the main PUFA in milk fat, but small amounts ofthe long chain PUFA, eicosapentaenoic acid (EPA; 20:5 n − 3) and docosahexaenoic acid(DHA; 22:6 n − 3) are also present. Generally, PUFA and MUFA are regarded as beneficialto human health and there is evidence of positive effects of trans-11 18:1 (the major transfatty acid in milk fat on most diets) in animal models (Corl et al., 2003; Lock et al., 2004),though other research suggests negative effects (Clifton et al., 2004). Milk fat is also amain dietary source of conjugated linoleic acid (CLA; Lawson et al., 2001; Ritzenthaler etal., 2001) for which an impressive list of health promoting biological properties have beenidentified in a range of biological models (Belury, 2002; Banni et al., 2003). More recently,consumption of CLA from dairy products was shown to be inversely related to colorectalcancer risk in the Swedish Mammography cohort in which records of over 60,000 womenwere analysed (Larsson et al., 2005). Lock and Bauman (2004) reported that 14 isomers ofCLA occur naturally in milk fat, but most interest has been focused on cis-9, trans-11 CLAand trans-10, cis-12 CLA for which biological effects have been investigated extensively.Their anti-mutagenic properties have been attributed to cis-9, trans-11 CLA (Ip et al., 1999;Corl et al., 2003; Lock et al., 2004), the main isomer of CLA in milk fat (Lock and Bauman,2004; Palmquist et al., 2005). However, milk fat trans-10, cis-12 CLA concentrations areextremely low (typically less than 0.01 g/100 g fatty acids) and, unless dietary supplementsare consumed, the intake of this isomer in most human diets would be negligible. Thisbackground has provided an impetus for research and for the dairy industry as a wholeto develop strategies to alter the composition of milk fat by decreasing its SFA content,particularly concentrations of 12:0, 14:0 and 16:0, and increasing, cis-9 18:1, n − 3 PUFAand CLA to be more compatible with recommendations for improving long-term humanhealth.

Manipulation of milk fat content, and its fatty acid composition, through breeding andnutrition strategies have been important targets for the dairy industry in many parts of theworld. Genetic advances have been particularly marked for level of fat, whereas nutri-tional approaches have had more distinct effects on fat composition (Lock and Shingfield,2004). A number of comprehensive reviews examining impacts of nutrition, including thesignificant role the rumen plays in the extensive metabolism of dietary lipids, on milkfatty acid composition have recently been published (Chilliard and Ferlay, 2004; Lock andBauman, 2004; Lock and Shingfield, 2004; Palmquist et al., 2005). The focus of this reviewis towards the role of forages in the diet on lipid metabolism in the rumen and their impor-tance to achieving strategic changes in milk fatty acid composition, particularly in relationto n − 3 PUFA and CLA, as well as reducing saturated fatty acids and increasing cis-9 18:1.Some consideration is given to trans 18:1 and 18:2 isomers, but information here is morelimited.

Forages provide a low-cost approach in comparison with diet supplementation strategies,such as oils and starch, which are designed to improve milk fatty acid profiles relativeto their impacts on the health of humans that consume them. Further, they do not resultin large increases in trans 18:1 isomers other than trans-11 18:1. Avoiding increases intrans isomers when trying to reduce SFA, and increase PUFA or CLA, is an importanttarget. Trans fatty acids are known to raise plasma total and LDL cholesterol concentrations


(Mensink and Katan, 1990), but in contrast to SFA, also lower high-density lipoprotein(HDL) cholesterol when substituted for cis unsaturated fatty acids (Zock and Katan, 1997;Williams, 2000). In response to concerns about impacts of consumption of trans fatty acidson cardiovascular risk, a number of countries have implemented, or plan to introduce,legislation to address these public health issues. It is mandatory to declare the trans fatcontent of foods in Canada and the United States. Since 2004, Denmark has implementedlegislation excluding use of oils and fats containing more than 0.02 trans fatty acids in themanufacture of processed foods. Other countries may soon introduce mandatory nutritionallabelling of the trans fat content of human foods (Ratnayake and Zehaluk, 2005). However,there is evidence to suggest that trans-9 and trans-10 18:1 are more potent in raising plasmaLDL cholesterol concentrations than trans-11 18:1 (Willett et al., 1993). Whilst furtherresearch is required to establish isomer specific effects, the profile of trans fatty acids variesamong foods. The distribution of trans 18:1 isomers is much more uniform in partiallyhydrogenated vegetable oils compared with milk fat in which trans-11 18:1 is typically themost abundant isomer (Molkentin and Precht, 1995; Lock and Shingfield, 2004; Lock etal., 2005; Table 1).

It is well established that consumption of trans fatty acids by humans is a risk factor forcoronary heart disease and diabetes (Ascheiro et al., 1999; Salmeron et al., 2001). However,recent data from a population based case–control study indicated that intake of trans 18:1was not associated with primary cardiac arrest risk, whereas consumption of trans, trans18:2 (trans 18:2) was associated with a three-fold increase in risk (Lemaitre et al., 2002). Theevidence emerging from clinical studies in humans suggests that trans fatty acids with morethan one trans double bond are particularly harmful. A number of reports have characterisedthe trans 18:2 content and profile of hydrogenated vegetable oils (Ratnayake, 2004), butinformation on the concentration and distribution of trans 18:2 in milk fat is limited (Kraft

Table 1Concentration of trans 18:1 fatty acids in edible fats (g/100 g total fatty acids)

Positionalisomer

Bovine milk fat Margarines Shortenings

Minimum Maximum Mean Minimum Maximum Mean Minimum Maximum Mean

18:1 trans-4 0.02 0.08 0.05 0.00 0.11 0.03 0.00 0.12 0.0318:1 trans-5 0.00 0.11 0.05 0.00 0.16 0.06 0.00 0.29 0.0818:1 trans-6,

-7 and -80.07 0.27 0.16 0.02 3.90 1.63 0.00 5.32 1.54

18:1 trans-9 0.16 0.30 0.23 0.05 4.84 2.04 0.01 6.52 2.2818:1 trans-10 0.03 0.30 0.17 0.04 5.30 1.93 0.01 6.74 1.9818:1 trans-11 0.35 4.43 1.72 0.03 4.50 1.38 0.01 5.19 1.4518:1 trans-12 0.10 0.31 0.21 0.02 4.08 1.12 0.00 4.06 1.1918:1 trans-13

and -140.00 0.85 0.49 0.01 3.56 0.92 0.00 3.25 0.97

18:1 trans-15 0.04 0.48 0.28 0.00 0.45 0.13 0.00 0.67 0.1818:1 trans-16 0.11 0.52 0.33 0.00 0.53 0.09 0.00 0.35 0.10

18:1 totaltrans

0.88 7.65 3.69 0.17 25.90 9.32 0.04 32.51 9.79

Adapted from Molkentin and Precht (1995).


et al., 2003; Shingfield et al., 2003, 2005a; Loor et al., 2005a). Furthermore, virtually allmeasurements of trans 18:2 concentrations in milk fat are based on the assumption thatisomers in milk fat are the same as those in partially hydrogenated vegetable oils. However,the trans 18:1 isomer profile is known to differ markedly between milk fat and partiallyhydrogenated vegetable oils (Table 1), and it is probable that this is also true for trans 18:2fatty acids.

Plants are the primary source of n − 3 fatty acids, whether in terrestrial or marine ecosys-tems. Developing strategies to exploit the potential of herbage PUFA as an alternative todwindling, and increasingly polluted, marine sources are important (Jacobs et al., 2004).Interrelationships between milk fatty acid composition and the processing and sensory char-acteristics of milk fat are not considered in this review, and the reader is directed to the recentreview by Chilliard and Ferlay (2004) for a more detailed appraisal.

2. Effects of conserved forages on milk PUFA

Considering the effects of conserved forages on beneficial milk PUFA, particularly CLAand 18:3 n − 3 fatty acids, factors affecting levels of precursor PUFA in forage will beaddressed first followed by forage effects on recovery of feed PUFA in milk.

2.1. Genetic and environmental effects on fatty acids in conserved forages

A number of researchers have investigated genetic variation in the levels of fatty acids ingrasses and legumes commonly consumed by ruminants. Fatty acid profiles can be used todifferentiate grass species and ryegrass families within cuts, but not among cuts (Dewhurstet al., 2001). Whilst these studies have provided some evidence of genetic effects, they haveoften identified large effects of environmental factors, such as light (Dewhurst and King,1998), cutting interval, season and fertiliser regime (discussed below). Apparent differencesamong species and cultivars must be treated with some caution, since most of these studieshave involved only a few cuts with no replication among growing seasons. Indeed, suchinteractions could mask genetic effects. For example Italian ryegrass had the highest levelsof fatty acids in vegetative material, but the lowest levels during the summer floweringperiod (Dewhurst et al., 2001). Boufaıed et al. (2003a) found the highest levels of fattyacids for annual ryegrass within grasses cut at the early heading stage, and for white cloverwithin legumes cut at the early bloom stage. However, there were other differences amongmanagement strategies within species. In addition, these authors found significant cultivareffects within species. Conversely, Elgersma et al. (2003a) found little difference amongperennial ryegrass cultivars when cut at a fixed herbage mass.

The number and timing of cuts within a year appears to have a major effect on the fattyacid concentrations in forages. A number of authors have demonstrated lower concentra-tions of fatty acids during summer (Bauchart et al., 1984; Dewhurst et al., 2001), whilstconcentrations may be maintained by intensive management that inhibits initiation of flow-ering (Dewhurst et al., 2002). Boufaıed et al. (2003a) and Dewhurst et al. (2001) foundlower concentrations for first-cut (‘Spring’) material than for regrowth (‘Summer’) grass,but the first cut material was cut at an advanced stage of maturity. This is consistent with


the general decline in concentrations of 18:3 n − 3 and total fatty acids with increasingregrowth interval (Table 2). Boufaıed et al. (2003a) found that use of N fertiliser increasedconcentrations of these fatty acids. The common basis for many of these effects appears tobe leaf/stem ratio with lower concentrations of fatty acids in stemmy regrowths (Bauchartet al., 1984; Elgersma et al., 2003b), although this relationship did not hold for herbage cutin autumn (Elgersma et al., 2003b).

2.2. Effects of conservation processes on fatty acids in forage

Oxidative loss of PUFA during field wilting represents a major loss in the food chain,with substantial losses of 18:3 n − 3 during hay making and modest losses during wiltingprior to ensiling. These losses are associated with the lipoxygenase system, a plant defencemechanism initiated in damaged tissues. Plant lipases release non-esterified 18:3 n − 3 and18:2 n − 6 from damaged membranes (Thomas, 1986) and these are rapidly converted tohydroperoxy PUFA by the action of lipoxygenases (Feussner and Wasternack, 2002). Thehydroperoxy PUFA are further catabolised to yield a range of volatile compounds, such asleaf aldehydes and alcohols (e.g., Fall et al., 1999). Table 3 shows the range of losses oftotal fatty acids and 18:3 n − 3 in a number of studies with different wilting conditions. Leaftissue contains more fatty acids than stem, so losses due to leaf shatter during hay makingwill also contribute to loss of fatty acids.

‘Stay-green’ grasses have provided one approach to reduce fatty acid losses during fieldwilting. We have investigated a ‘stay-green’ grass which lacks one of the enzymes involvedin chlorophyll breakdown (Thomas and Smart, 1993) and so retains a thylakoid membranestructure later in senescence. Stay-green grasses had a substantial retention of fatty acidswhen artificially senesced (i.e., kept on moist filter paper in the dark; Harwood et al., 1982),but there was little effect on reducing substantial fatty acid losses during artificial wilting(Dewhurst et al., 2002).

Steele and Noble (1983) showed that there was considerable hydrolysis of the majorfatty acid containing fractions (i.e., galacto- and phospholipid) in grass during ensiling,with a concomitant increase in levels of free fatty acids. However, they found no evidencefor interconversion of fatty acids during silage fermentation.

A number of studies, at a range of scales (Dewhurst and King, 1998; Boufaıed et al.,2003a; Shingfield et al., 2005b) have shown significantly higher levels of total fatty acidsand 18:3 n − 3 in grass silages prepared with formic acid additive. The basis of this effectis not clear, though it may relate to differential loss of other dry matter (DM) during silagefermentation. One possible mechanism for effects of formic acid is an effect on effluentlosses (e.g., Kennedy, 1990). However effluent losses did not occur in Dewhurst and King(1998).

2.3. Species effects on transfer of forage PUFA into milk

Recent studies (Dewhurst et al., 2003a; Al-Mabruk et al., 2004; Tables 4 and 5) showedsignificantly increased levels of 18:3 n − 3 and 18:2 n − 6 in milk when cows were fedred clover silage versus grass silage. Apparent recoveries of 18:3 n − 3 from feed to milkincreased from about 0.04 to 0.05 for grass silage-based diets to about 0.08–0.10 for diets

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Table 2Effects of growth duration on total fatty acids (g/kg DM) and �-linolenic acid (18:3 n − 3; g/kg DM) in grasses

Reference Grassspecies

Dates of primary growth orregrowth interval (days)

Total fatty acids 18:3 n − 3

Shortregrowth

Longregrowth

Significant Shortregrowth

Longregrowth

Significant

Dewhurst et al. (2001) Lolium perenne 20 days vs. 38 days 24.7 17.5 P<0.01 16.4 10.9 P<0.01Dewhurst et al. (2001) Lolium multiflorum 20 days vs. 38 days 25.3 12.9 P<0.01 17.3 7.5 P<0.01Boufaıed et al. (2003a) Phleum pratense Primary growth at 2

or 23 June19.8 15.3 P<0.01 10.1 6.9 P<0.01

Elgersma et al. (2003a) L. perenne 23 days vs. 33 days 26.2 22.6 NS 17.8 14.5 NS

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Table 3Effects of wilting on total fatty acids (g/kg DM) and �-linolenic acid (18:3 n − 3; g/kg DM) in herbage at ensiling (fresh grass vs. silage comparison for Elgersma et al.,2003a)

Reference Grass species Description of wilt Total fatty acids 18:3 n − 3

Unwilted Wilted Significant Unwilted Wilted Significant

Dewhurst and King (1998) L. perenne 68 h in lab 24.8 19.4 P<0.001 13.4 8.9 P<0.001Dewhurst et al. (2002) L. perenne 18 h (48 h) in lab 29.4 23.1 (21.1) P<0.001 17.9 13.9 (12.7) P<0.001Boufaıed et al. (2003a) P. pratense A few hours in field 19.3 16.6 P<0.01 9.3 8.1 P<0.01Elgersma et al. (2003a) L. perenne 24 h in glasshouse 29.1 19.5 P<0.001 19.9 12.6 P<0.001

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Table 4Effects of clover silages on concentrations of unsaturated fatty acids in milk (g/100g total fatty acids)Reference Clover species and

concentrate feedinglevel (kg/day)

18:3 n − 3 18:2 n − 6 CLA Trans-11 C18:1 Total C18:1 (cis-9 and cis-11)

Grass Clover Significant Grass Clover Significant Grass Clover Significant Grass Clover Significant Grass Clover SignificantDewhurst et al.

(2003a,b) (1)T. repens (8) 0.43 0.91 P<0.001 1.42 1.80 P<0.01 Not stated Not stated Not stated Not stated Not stated Not stated 24.7 23.0 NS

Dewhurst et al.(2003a,b) (2)

T. repens (8) 0.40 0.96 P<0.001 1.05 1.54 P<0.001 0.36 0.34 NS 1.13 1.06 NS (20.7) (17.9) P<0.05


T. pratense (8) 0.43 0.81 P<0.001 1.42 1.81 P<0.01 Not stated Not stated Not stated Not stated Not stated Not stated 24.7 24.0 NS


T. pratense (8) 0.40 1.28P<0.001

1.05 1.58P<0.001

0.36 0.41P<0.05

1.13 1.25P<0.05

20.7 20.2 NST. pratense (4) 0.48 1.51 0.90 1.47 0.37 0.42 1.16 1.31 19.0 20.0

Al-Mabruk et al.(2004)

T. pratense (8) 0.48 0.92 P<0.05 1.24 1.54 P<0.001 0.45 0.40 P<0.001 1.31 1.16 P<0.01 23.2 23.5 NS

NS, not significant (P>0.05).

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Table 5Effects of clover silages on concentrations of saturated fatty acids in milk (g/100 g total fatty acids)Reference Clover species and

concentrate feedinglevel (kg/day)

12:0 14:0 16:0 18:0

Grass Clover Significant Grass Clover Significant Grass Clover Significant Grass Clover SignificantDewhurst et al. (2003a,b) T. repens (8) 4.26 4.73 NS 12.2 13.0 NS 31.5 29.7 NS 10.5 9.9 NSDewhurst et al. (2003a,b) T. repens (8) 3.52 4.16 P<0.05 11.7 12.7 P<0.05 32.5 32.9 NS 11.0 9.7 P<0.001Dewhurst et al. (2003a,b) T. pratense (8) 4.26 4.28 NS 12.2 12.2 NS 31.5 31.2 NS 10.5 10.1 NS

Dewhurst et al. (2003a,b)T. pratense (8) 3.52 3.31

P<0.0511.7 11.3

P<0.0532.5 30.6

P<0.00111.0 11.6

P<0.05T. pratense (4) 3.34 2.89 12.0 10.9 34.3 30.6 10.7 11.6

Al-Mabruk et al. (2004) T. pratense (8) 3.06 2.89 NS 10.6 10.5 NS 31.0 30.5 NS 12.6 11.9 NSNS, not significant (P>0.05).


based on red clover silage. The highest level of 18:3 n − 3 in milk (i.e., 1.51 g/100 g totalfatty acids) occurred when red clover silage was fed with only 4 kg/day of concentrates.White clover silage caused higher levels of 18:3 n − 3 fatty acid in milk versus milk fromgrass silage-based diets in both studies of Dewhurst et al. (2003a). This resulted both fromhigher levels of 18:3 n − 3 in white clover silage, and a slightly higher recovery from feedto milk. There were no consistent effects of red or white clover silages on saturated fattyacids, cis-9 18:1, cis-9, trans-11 CLA or trans-11 18:1 in milk fat.

Adesogan et al. (2004) and Salawu et al. (2002) compared a series of pea–wheat bi-cropsilages with grass silages on milk production. There were no effects of pea–wheat bi-cropsilages on cis-9, trans-11 CLA or trans-11 18:1, although 16:0 generally increased withthis forage and cis-9 18:1 decreased. Levels of 18:3 n − 3 in milk were similar amongtreatments. However, Salawu et al. (2002) showed that recovery of 18:3 n − 3 from feedin milk increased from 0.066 for grass silage to 0.086 and 0.108 for early- and late-cutbi-crops, respectively.

Maize silage contains only low levels of 18:3 n − 3 and much higher levels of cis-9 18:1.Consequently, maize silage-based diets resulted in milk with lower levels of beneficialPUFA than milk from cows fed winter oat pasture (Schroeder et al., 2003), pasture (Regoet al., 2004) or grass/clover silage (Havemose et al., 2004). Chilliard and Ferlay (2004)reported unpublished data showing higher levels of 18:3 n − 3, but similar levels of CLA,in milk from cows fed diets based on grass silage versus diets based on maize silage. Theyalso reported results from research in which levels of CLA and trans-11 18:1 were similarin goats fed maize silage or lucerne hay. Schroeder et al. (2003) and Rego et al. (2004)showed increased saturated fatty acids, particularly 16:0, and decreased cis-9 18:1 whenfeeding maize silage-based total mixed rations.

2.4. Effects of conservation methods on transfer of feed PUFA into milk

Shingfield et al. (2005b) compared fatty acid profiles of milk produced from hay orsilages made from a mixture of Timothy and Meadow fescue, and found that there were nooverall effects on SFA or MUFA. Cows offered hay produced more 18:3 n − 3 in milk thancows fed silages, despite having about half the intake of 18:3 n − 3. There was no overalldifference in levels of CLA or trans-11 18:1 in milk produced from hay or silage. Theapparent transfer of 18:3 n − 3 from diet into milk increased from 0.033 for grass silage-based diets to 0.172 for diets based on grass hay. Although feed information is inadequate tocalculate recoveries from feed to milk or biohydrogenation, it is clear that recoveries weremuch higher for diets based on very mature hay versus diets based on fresh grass or haylagein the studies described by Aii et al. (1988). Similarly, Chilliard et al. (2001) concluded thatlevels of 18:3 n − 3 in milk from cows offered grass hay were much higher than expectedin the studies of Decaen and Adda (1970) and Bartsch et al. (1979). This observation isconsistent with reduced 18:3 n − 3 biohydrogenation in vitro for Timothy hay versus withfresh or wilted grass, haylage or silage (Boufaıed et al., 2003b).

Shingfield et al. (2005b) showed increased levels of CLA and trans-11 18:1, and reducedlevels of 18:3 n − 3, in milk when they used a high rate (i.e., 8.3 L/t) of formic acid-basedproduct rather than an inoculant/enzyme preparation as silage additive, although they didnot claim it as a consistent effect.


3. Effects of grazed herbage on milk PUFA

Grazed herbage generally leads to increased levels of PUFA in milk. For example,Chilliard et al. (2001) referred to concentrations of 18:3 n − 3 in milk of up to 0.032 g/g ofmilk fatty acids in pastured cows. A number of studies have compared milk fatty acid pro-files from grazing cows to milk of cows fed hay or silage-based diets (Tables 6 and 7). Thesestudies range from surveys comparing milk produced from pasture during the grazing seasonwith milk produced from conserved forages with housed feeding to more controlled compar-isons, although it is obviously not possible to compare fresh forage with material conservedfrom the same forage at the same time. In fact, comparisons are further complicated by differ-ences in the forage:concentrate (F:C) ratio for cows fed silage-based rations versus pasture.It is not possible to calculate F:C ratios for early studies, whilst pastured cows receivedproportionately less concentrate than those fed total mixed rations (TMR) in the studies ofKelly et al. (1998; forage comprised 0.47 and 1.00 of TMR and pasture diets, respectively)and White et al. (2001; forage comprised 0.59 and approximately 0.75 of TMR and pasturediets, respectively). Only in the study of Offer (2002) was concentrate feeding level low(3 kg/day) and a relatively fixed proportion of DM intake (0.87 and 0.85, respectively).

Whilst effects of fresh forage on increasing 18:3 n − 3 and CLA are consistent among anumber of studies, there are no consistent effects on other fatty acids—perhaps because ofdifferences in the level and type of concentrates offered. A few comparisons have been madewith other grazed species. For example, Schroeder et al. (2003, 2005) reported increasedconcentrations of 18:3 n − 3 and CLA with oat pasture versus a silage-based TMR, whilstWhiting et al. (2004) showed increased concentrations of 18:3 n − 3 and 18:2 n − 6 in milkfrom fresh (wilted) versus ensiled lucerne. The increasing concentrations of 18:2 n − 6 inmilk from cows fed conserved forage in the studies of Kelly et al. (1998), Dhiman et al.(1999), White et al. (2001) and Schroeder et al. (2003) probably reflect increased levels of18:2 n − 6-rich concentrate in these diets. This illustrates the difficulty of identifying forageeffects on concentrations of 18:2 n − 6 in milk in most studies.

3.1. Fatty acid concentrations in grazed herbage

Effects of forage species and cultivar described above for conserved forages are alsorelevant to grazed pasture. Indeed, genetic differences in the fatty acid concentrations willbe more apparent in young growing plants, versus when flowering and senescence becomeimportant in more mature grasses destined for conservation as silage or hay.

Auldist et al. (1998), Mackle et al. (1999) and Thomson and Van Der Poel (2000)studied milk fatty acids in the New Zealand pasture based system of milk production, andshowed that concentrations of PUFA and CLA in milk fat were highest during spring andautumn with declines during summer that reflected reduced pasture quality. There were alsoreductions in MUFA, and increased SFA, during summer. Lock and Garnsworthy (2003)showed similar effects under UK conditions in pasture systems. Nudda et al. (2005) reportedthe fatty acid profiles of milk and dairy products from sheep grazing pasture, and foundthat concentrations of 18:3 n − 3, cis-9, trans-11 CLA and trans-11 18:1 all declined, byproportionately 0.36, 0.48 and 0.61, respectively, between spring and summer. The authorsattributed this effect to declining quality and quantity of pasture.

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Table 6Comparison of the effects of fresh pasture and conserved forages on concentrations of unsaturated fatty acids in milk (g/100 g total fatty acids)Reference Conserved

forage18:3 n − 3 18:2 n − 6 CLA Total 18:1 (trans-11 18:1)

Freshherbage

Conservedforage

Significant Freshherbage

Conservedforage


Conservedforage


Conservedforage

Significant

Timmen and Patton(1988)

Grass or wheatsilage

0.84 0.36 Not stated 2.20 2.27 Not stated 1.34 0.27 Not stated 25.2 17.0 Not stated

Aii et al. (1988) Grass hay 1.65 1.29 P<0.01 2.34 2.51 NS Not stated Not stated Not stated 26.6 31.3 P<0.01Precht and

Molkentin(1997)

Grasssilage/maizesilage/hay

Not stated Not stated Not stated 1.17 1.29 Not stated 1.20 0.45 Not stated Not stated Not stated Not stated

Kelly et al. (1998) Maizesilage/legumesilage/ legumehay

0.95 0.25 P<0.01 2.25 2.62 P<0.05 1.09 0.46 P<0.01 34.7 26.6 P<0.01

White et al. (2001) Maizesilage/lucernesilage

0.71 0.38 P<0.01 1.82 2.49 P<0.01 0.72 0.41 P<0.01 23.1 23.3 NS

Offer (2002) Grass silage 0.76 0.41 Not stated 0.85 0.71 Not stated 2.64 0.68 Not stated (3.86) (0.94) Not statedNS, not significant (P>0.05).

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Table 7Comparison of the effects of fresh pasture and conserved forages on concentrations of saturated fatty acids in milk (g/100 g total fatty acids)Reference Conserved

forage12:0 14:0 16:0 18:0

Freshherbage

Conservedforage


Conservedforage


Conservedforage


Conservedforage

Significant

Timmen and Patton (1988) Grass or wheatsilage

3.43 4.80 Not stated 10.4 12.6 Not stated 26.6 35.4 Not stated 10.0 6.5 Not stated

Aii et al. (1988) Grass hay 3.22 2.36 P<0.01 10.8 8.2 P<0.01 27.2 26.6 NS 10.4 10.8 P<0.05Kelly et al. (1998) Maize

silage/legumesilage/ legume hay

1.70 2.59 P<0.01 6.7 9.4 P<0.01 24.2 30.7 P<0.01 13.2 15.0 P<0.05

White et al. (2001) Maizesilage/lucernesilage

2.65 2.34 P<0.01 10.2 9.4 P<0.01 31.2 31.7 NS 13.4 15.4 P<0.01



Table 8Effects of varying pasture allowance on concentrations of �-linolenic acid (18:3 n − 3) and CLA in milk (g/100 gtotal fatty acids)

Reference Pasture allowance(kg DM/day)

18:3 n − 3 CLA

Lowallowance

Highallowance

Significant Lowallowance

Highallowance

Significant

Stanton et al.(1997)

16 vs. 24 ND ND 0.39 0.57 P<0.05

Wales et al.(1999)

20 vs. 70 0.6 0.6 NS 1.4 1.4 NS

Stockdale etal. (2003)

25 vs. 50 0.6 0.5 NS 1.2 1.5 P<0.05

Stockdale etal. (2003)

25 vs. 50 0.6 0.6 NS 1.05 1.05 NS


Effects of pasture allowance on milk PUFA are summarised in Table 8. There is muchgreater potential for selection of material in a grazing situation, and so feeding methods (e.g.,zero-grazing) and methods of allocating pasture could also influence milk fatty acid profiles.Pasture allowance could exert an effect on milk fatty acid profiles through the compositionof pasture consumed by forcing cows to graze into lower quality herbage at the base of thesward. Care must be taken to distinguish increased pasture allowance achieved by alteringland area at a given pasture mass versus by altering pasture mass per unit area, whichprobably means altering plant growth stage. Wales et al. (1999) separated effects of pasturemass, and compared widely different pasture allowances, finding no significant effects on18:3 n − 3 (mean = 0.006 g/g total fatty acids) or cis-9, trans-11 CLA (mean = 0.014 g/gtotal fatty acids) in milk.

Effects of pasture on milk fatty acid levels are reflected, to a lesser extent, in effects ofpartial inclusion of pasture in the diet (Kelly et al., 1998). Access to pasture for just 8 h ledto significant increases in concentrations of 18:3 n − 3, cis-9, trans-11 CLA and trans-1118:1 for cows offered a total mixed ration (Loor et al., 2003). Dhiman et al. (1999) showedparallel increases in 18:3 n − 3 and cis-9, trans-11 CLA by increasing pasture allowancefrom one-third to all of diets.

3.2. Transfer of herbage PUFA into milk

Grazing birdsfoot trefoil (Lotus corniculatus) versus perennial ryegrass led to increasedconcentrations of 12:0, 14:0, 16:0, 18:2 n − 6 and 18:3 n − 3, and reduced concentrationsof cis-9 18:1, cis-9, trans-11 CLA and trans-11 18:1 fatty acid in milk (Turner et al., 2005).Addition of polyethylene glycol, which blocks the action of tannins in the digestive tract,showed that the effect on 18:3 n − 3 (i.e., reduced biohydrogenation) was almost certainlyrelated to the effects of Lotus tannins, whilst effects on biohydrogenation intermediates,notably a reduced trans-11 18:1 concentration, were probably related to some other Lotuscomponent. Studies with the tannin-containing legume Sulla fed to lactating sheep reachedsimilar conclusions. Piredda et al. (2002), cited by Addis et al. (2005), showed increased


18:3 n − 3 in milk when comparing Sulla pasture with perennial ryegrass at either vegetativeor reproductive stages. There were no consistent effects on trans-11 18:1 or cis-9, trans-11CLA. Addis et al. (2005) demonstrated similar effects of Sulla in both winter and spring.Grazing Sulla also led to a large increase in 12:0 (4.59 g/100 g versus 6.52 g/100 g total fattyacids) and a large reduction in cis-9 18:1 (22.0 g/100 g versus 13.3 g/100 g total fatty acids).Interestingly, these studies also identified an herb (i.e., Chrysanthemum coronarium) whichled to consistently higher concentrations of cis-9, trans-11 CLA, and trans-11 18:1 andlower concentrations of 12:0 and 16:0, in milk than from sheep grazing annual ryegrass.There is preliminary evidence (R.J. Dewhurst, unpublished) for increases in 18:3 n − 3concentrations in milk from cows grazing either white or red clover in comparison withryegrass, though effects appear smaller than with red clover silage.

Delagarde and Peyraud (2002) and Loyola et al. (2002) presented preliminary evidencefor modest effects of grass species and cultivars on 18:3 n − 3, CLA and trans-11 18:1 inmilk. However, longer-term results are needed to determine if effects are real because of thelarge potential for Genotype×Environment interactions in forage composition. It remainsto be seen whether any of these preliminary observations relate to real species and cultivareffects on milk PUFA.

Offer (2002) compared milk from cows grazing pasture with milk from cows offered cutmaterial from the same field. Zero-grazing led to a small (proportionately 0.24) reduction inmilk 18:3 n − 3 proportion, but a much larger reduction in cis-9, trans-11 CLA and trans-1118:1 (proportionately 0.52). It has been suggested that some of this effect may be due to agreater potential by cattle to select high-quality pasture in the field. This may explain thereduction in 18:3 n − 3, but clearly the effect on biohydrogenation intermediates was higherand some other mechanism is probably involved.

A number of studies have identified elevated concentrations of CLA and n − 3 fattyacids in Alpine milk and dairy products (comparisons are summarised in Tables 9 and 10).Interpreting the causes of these effects must be done with care since there are a numberof differences among production systems. Nonetheless, the consistency of effects in thesesurveys suggests that the area merits further attention. Leiber et al. (2005) showed increasedconcentrations of 18:3 n − 3 in Alpine milk despite the Alpine pasture containing less 18:3n − 3 than lowland forage, which was dominated by ryegrass and white clover. Alpine milkalso generally contains less medium-chain saturated fatty acids and more 18:1, both cisand trans. As with fresh lowland pasture, effects on 18:2 n − 6 were inconsistent, probablybecause of much higher variation in supply of 18:2 n − 6 from concentrates. In this regard,studies of 18:3 n − 3 are less confounded because 18:3 n − 3 is usually a much smallercomponent of concentrates than is 18:2 n − 6.

Table 11 provides a summary of the effects of forage treatments on concentrations ofimportant fatty acids in milk. Dietary 18:3 n − 3 is the ultimate precursor of 18:3 n − 3 inmilk, whilst CLA (cis-9, trans-11) derives from dietary 18:2 n − 6 and 18:3 n − 3. Theseare the two major fatty acids in forages and their concentrations are usually closely corre-lated. Consequently, it is not surprising that some of the variation in milk PUFA profilesrelates to concentrations of these fatty acids in forage. However, despite their commonprecursors, Table 11 shows that it is possible to alter concentrations of 18:3 n − 3 andCLA in milk independently. This suggests that some other variation in milk PUFA resultsfrom effects of forages on rates of lipolysis and changes in the relative importance of spe-

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Table 9Effects of Alpine pasture on concentrations of unsaturated fatty acids in milk (g/100 g total fatty acids)Reference Study details 18:3 n − 3 18:2 n − 6 CLA Trans-10 and11 18:1 (trans-11) cis-9 18:1 (total 18:1)

Lowland Alpine Significant Lowland Alpine Significant Lowland Alpine Significant Lowland Alpine Significant Lowland Alpine SignificantHebeisen et al.

(1993)Milk from cows onpasture at1000–1500 m a.s.l.vs. milk fromlowland cows ongrass silage andmaize silage

0.45 2.31 P<0.01 1.77 2.55 NS Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated

Collomb et al.(2001,2002)

Milk from cowsgrazing at600–650 m a.s.l. or1275–2120 m a.s.l.

0.79 1.15 P<0.05 1.14 1.33 P<0.01 0.81 2.18 P<0.05 2.11 5.10 P<0.05 16.7 17.4 NS

Innocente etal. (2002)

Cheese from Alpinesummer pasture vs.cheese from cowsfed conservedforage and grain

0.50 1.10 P<0.001 2.84 2.50 P<0.01 0.75 1.63 P<0.001 Not stated Not stated Not stated (25.3) (33.8) P<0.001

Kraft et al.(2003)

Milk from Alpinesummer pasture at>1200 m a.s.l. vs.milk from cows fedsilage andconcentrates at200 m a.s.l.

0.33 1.17 P<0.05 1.63 1.19 P<0.05 0.35 3.43 P<0.05 (0.33) (3.23) P<0.05 18.2 20.6 P<0.05

Hauswirth etal. (2004)

Cheese from cowsgrazing at1150–1900 m a.s.l.vs. English cheddarcheese

0.6 1.5 P<0.001 Not stated Not stated Not stated 0.9 2.5 P<0.001 Not stated Not stated Not stated Not stated Not stated Not stated

Leiber et al.(2005)

Milk from cowsgrazing pasture at2000 m a.s.l. vs.milk from matchedcows fedsilage/hay/concentratesat 400 m a.s.l.

0.54 1.15 P<0.001 1.31 1.57 P<0.05 0.55 1.34 P<0.001 0.73 3.12 P<0.001 20.2 24.1 P<0.001

m a.s.l., metres above sea level; NS, not significant (P>0.05).

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Table 10Effects of Alpine pasture on concentrations of saturated fatty acids in milk (g/100 g total fatty acids)Reference Study details 12:0 14:0 16:0 18:0

Lowland Alpine Significant Lowland Alpine Significant Lowland Alpine Significant Lowland Alpine SignificantHebeisen et al.

(1993)Milk from cows onpasture at1000–1500 m a.s.l.vs. milk fromlowland cows ongrass silage andmaize silage

Not stated Not stated Not stated Not stated Not stated Not stated 46.1 41.1 NS Not stated Not stated Not stated

Collomb et al.(2001,2002)

Milk from cowsgrazing at600–650 m a.s.l. or1275–2120 m a.s.l.

2.63 2.13 P<0.05 9.0 7.9 P<0.05 24.1 20.8 P<0.05 9.6 9.0 P<0.05

Innocente etal. (2002)

Cheese from Alpinesummer pasture vs.cheese from cowsfed conservedforage and grain

3.46 2.47 P<0.001 11.7 8.8 P<0.001 30.5 23.5 P<0.001 9.8 10.7 NS

Kraft et al.(2003)

Milk from Alpinesummer pasture at>1200 m a.s.l. vs.milk from cows fedsilage andconcentrates at200 m a.s.l.

3.35 2.21 P<0.05 9.5 7.9 P<0.05 27.3 20.7 P<0.05 7.5 9.8 P<0.05

Hauswirth etal. (2004)

Cheese from cowsgrazing at1150–1900 m a.s.l.vs. English cheddarcheese

30.1 24.7 P<0.001

Leiber et al.(2005)

Milk from cowsgrazing pasture at2000 m a.s.l. vs.milk from matchedcows fedsilage/hay/concentratesat 400 m a.s.l.

3.00 2.09 P<0.001 10.8 8.5 P<0.001 33.7 25.4 P<0.001 9.4 11.8 P<0.001

m a.s.l., metres above sea level; NS, not significant (P>0.05).

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Table 11Summary of the effects of different forage treatments on fatty acid concentrations in milk fat

Forage treatment 18:3 n − 3 CLA Trans-11 18:1 Cis-9 18:1 12:0, 14:0 and 16:0 18:0

Fresh herbage (vs. silage) Increases Increases Increases – – –Leafy (Spring) pasture Increases Increases Increases Increases Decreases –Increasing pasture allowance – – – – – –Alpine pasture (vs. lowland diet) Increases Increases Increases Increases Decreases –Grazing birdsfoot trefoil (vs. grass) Increases Decreases Decreases Decreases Increases DecreasesGrazing Sulla (vs. grass) Increases – – Decreases Increases 12:0 –Grazing Chrysanthemum coronarium (vs. grass) – Increases Increases – Decreases –Zero-grazing (vs. grazing) Decreases Decreases Decreases – – –Hay (vs. silage) Increases – – – – –Formic acid additive on silage Decreases Increases – – – –Red clover silage (vs. grass silage) Increases – – – – –White clover silage (vs. grass silage) Increases – – – – –Whole-crop pea/wheat silage (vs. grass silage) – – – Small decrease Increases 16:0 –Maize silage TMR (vs. pasture) Decreases Decreases Decreases Decreases Increases Decreases

(–) no consistent effect within the available literature.


cific metabolic steps of ruminal biohydrogenation. Section 4 describes several aspects ofplant chemical composition and biochemistry which may be involved in some of theseeffects.

4. Plant mechanisms affecting ruminal lipolysis and biohydrogenation

4.1. Rumen retention time

There is increasing evidence that not all forages behave in the same way when exposedto biohydrogenating bacteria. Studies in dairy cows (Dewhurst et al., 2003b) and steers(Lee et al., 2003a) demonstrated more extensive ruminal biohydrogenation of 18:3 n − 3 ingrass silage compared to white or red cover silage. Dewhurst et al. (2003b) suggested thatthe reduced biohydrogenation with white clover silage may be due to much higher ruminalpassage kinetics that reduces exposure of forage lipids to lipases and biohydrogenation inthe rumen. Increased passage rates from the rumen with whole-crop cereal silage (Abdallaet al., 1999) may have contributed to increased recovery of 18:3 n − 3 from pea–wheatsilage to milk.

4.2. Effects on lipolysis

Other mechanisms affecting ruminal biohydrogenation appear to operate through effectson lipolysis. Lipolysis of ester linkages of dietary lipids is the initial step in lipid metabolismin the rumen. Products liberated during lipolysis of triacylglycerols or associated cell andorganelle membrane lipids (mono- and di-galactosyl glycerol, sulpholipid, phosphatidyl-choline, phosphatidylglycerol, phosphatidylethanolamine) are rapidly metabolized to yieldnon-esterified fatty acids (Harfoot and Hazelwood, 1988; Palmquist et al., 2005). The extentof lipolysis of unprotected oils has been estimated to be in the range of 0.85–0.95, whilethe hydrolysis of structural plant lipids is thought to be lower due to the need to remove sur-rounding cellular matrices before lipolysis can occur (Doreau and Ferlay, 1994). Lipolysisof esterified lipids in the diet is considered to be rate limiting for biohydrogenation (Harfootand Hazelwood, 1988). Early experiments provided evidence to suggest that lipolysis wouldbe sufficiently rapid to allow the hydrogenating capacity of rumen microbes to be realized(Hawke and Silcock, 1970). However, further studies have shown that the rate of lipolysisin vitro is altered by diet composition, being more extensive in response to increases inthe N content of high starch diets (Gerson et al., 1983) and decreased when dietary fibre isreplaced with starch (Latham et al., 1972; Gerson et al., 1985). Lipolysis in vitro has beenreported to be decreased by advances in forage maturity (Gerson et al., 1986), and thereis evidence that drying, versus ensiling, also reduces the extent of hydrolysis of foragelipids (Boufaıed et al., 2003a,b). Furthermore, lipolysis of meadow hay was shown to bedependent on particle size, being higher during incubation with forage particles of between1 and 2 mm versus 0.1–0.4 mm in size (Gerson et al., 1988). Although in vitro studies haveshown that rate and extent of lipolysis is dependent on a number of factors related to dietcomposition, demonstrating that these effects occur in vivo has proven to be much moredifficult.


Red clover contrasts to white clover in that it has a similar fibre content and rumen passagerate versus grass (Dewhurst et al., 2003b) and its effect on the extent of biohydrogenationappears to be related to differences in inherent plant enzymatic activity. Plant lipases presentin the leaves of pasture plants have been shown to remain active for at least 5 h in the pres-ence of metabolizing rumen micro-organisms (Faruque et al., 1974) and may be responsiblefor the first stages of lipolysis when ruminants graze fresh pasture (Lee et al., 2002). Redclover has a much lower lipolytic activity than grass (Lee et al., 2003b) due to the effect ofthe enzyme polyphenol oxidase (PPO; Lee et al., 2004). Recent studies from the Institute ofGrassland and Environmental Research (M.R.F. Lee, L.J. Parfitt and F.R. Minchin, unpub-lished results) suggest that effects of PPO involve protection of plant lipids from lipolysisas well as denaturation of plant lipases. Forage lipids are mainly in the form of polar mem-brane lipids which form complexes with the highly electrophilic o-quinones produced byPPO. It is suggested that these polar lipid–phenol complexes may offer some protectionfrom lipolysis and hence result in PUFA escaping rumen biohydrogenation. To date, effectsof PPO in red clover on lipolysis has been demonstrated in red clover silage and with invitro incubations of macerated material but, as yet, there is little evidence about the effectof polyphenol oxidase in grazing cattle. The enzyme requires the presence of oxygen andso would not be expected to operate within the anaerobic rumen. However, PPO acts withinseconds and has been shown to be activated during the cell damage associated with ingestionand mastication, meaning that the enzyme may still have an effect during grazing of freshred clover (M.R.F. Lee, A.L. Winters, R.J. Dewhurst and F.R. Minchin, unpublished results).Recent studies have found PPO in some grasses (Marita et al., 2005; Lee et al., 2006).

Leiber et al. (2005) suggested that the Alpine effect on milk fatty acids (Tables 9 and 10)may be due to preferential mobilisation of 18:3 n − 3 from body reserves when cows aregrazing these pastures at high altitude, and/or reduced biohydrogenation as a consequenceof one or more plant components from the diverse range of species found at these altitudes.This hypothesis requires further evaluation. These authors noted the presence of condensedtannin-rich legumes and red clover, but other species could contain compounds such assaponins (Shi et al., 2004), proanthocyanidins (Moreno et al., 2003) and catecholamines(Lafontan et al., 2002), and all been shown to inhibit lipases.

4.3. Effects on biohydrogenating bacteria

One of the simplest approaches to reduce rumen biohydrogenation is to alter the rumenmicroflora by reducing rumen pH. This effect has been achieved in a number of studies withdiets containing a high proportion of starch-rich concentrates (e.g., Kalscheur et al., 1997a;Kucuk et al., 2001; Piperova et al., 2002). Generally this mitigates against increasing milkPUFA in high-forage systems since forages usually lead to a relatively high rumen pH.Condensed tannins, such as those in L. corniculatus and Sulla, provide another approach toreducing biohydrogenation. In this case, the mechanism appears to be selective inhibitionof several strains of Butyrivibrio fibrisolvens (Min et al., 2003), one of the most importantbiohydrogenating ruminal bacteria species.

Several studies (Table 6) have shown increased concentrations of 18:3 n − 3 and CLA inmilk produced from cows grazing pasture versus those consuming diets based on conservedforages such as maize silage, grass silage or hay. This ‘pasture effect’ may be due to the


different fatty acid containing fractions of fresh grass and conserved forage (Steele andNoble, 1983). Lee et al. (in press) reported that about 0.70 of PUFA in fresh forage was inthe form of polar membrane lipid, 0.15 as diacylglycerol, 0.08 as triacylglycerol and only0.07 were as NEFA, whereas in silage about 0.28 of PUFA was in the polar membrane lipid,0.09 as diacylglycerol, 0.20 as triacylglycerol and 0.43 as NEFA. However, Offer (2002)showed that a similar depression in milk fat CLA could be achieved if the grass was cut andfed after a short wilt, which suggests that the change was rapid and not necessarily relatedto the form of PUFA containing lipid in fresh and ensiled grass. Morphological differencesbetween the relatively turgid fresh and flaccid wilted grass were investigated by Lee et al.(2005a), who reported that wilted grass still resulted in lower concentrations of CLA andtrans 18:1 formation in vitro, even when both wilted and unwilted grass were homogenisedto remove morphological differences.

The earlier section discussed the involvement of plant lipoxygenases in losses of PUFAduring field wilting. It is now becoming apparent that these processes may also have effectson rumen function. Hatanaka (1993) and de Gouw et al. (1999) reported the compositionof the volatile compounds released during cutting of grass. This ‘green odour’ consistsof the products of fatty acid oxidation completed by plant lipoxygenases. These enzymescatalyse oxygenation of PUFA to form a mixture of volatile hydroperoxides, alcohols,aldehydes and ketones. These compounds have antimicrobial functions in nature (Kubo etal., 1995) and may have an effect on rumen microbial populations. This ‘green odour effect’may offer a partial explanation for differences in the fatty acid composition of milk frompastured cows versus those fed conserved forages. Preliminary studies (Lee et al., 2005b)have demonstrated effects of two typical fatty acid oxidation products (i.e., hydroperoxidesand long chain aldehydes) in increasing biohydrogenation of 18:2 n − 6 and 18:3 n − 3, aswell as increasing formation of trans-11 18:1 in vitro.

5. Effects of dietary forage: concentrate ratio on ruminal biohydrogenation

There is increasing evidence that in addition to forage species and conservation method,the relative proportion of forage in the diet is an important factor regulating the extentof ruminal biohydrogenation and the formation of fatty acid intermediates. Reducing theF:C ratio of diets containing maize silage and lucerne haylage from 60:40 to 25:75 (DMbasis) increased flow of trans 18:1 intermediates from the rumen, effects that were alleviatedby including buffers in the diet (Kalscheur et al., 1997a). More detailed analysis revealedthat flow of specific biohydrogenation intermediates was also altered by changes in theproportion of forage in the diet (Piperova et al., 2002; Table 12). Further research hasestablished that similar changes in flow of fatty acids at the duodenum also occurs incows fed rations based on grass hay when the F:C is decreased from 65:35 to 35:65 (DMbasis; Loor et al., 2004; Table 12). Overall, most evidence suggests that increases in theproportion of concentrates in the diet can be expected to increase the amounts of trans-10, 13 and 14, and 15 18:1, 18:2 n − 6 and total fatty acids leaving the rumen. Consistentwith the findings in dairy cows, changing from dehydrated grass pellets to high concentratediets substantially increased amounts of trans-10 18:1 in abomasal digesta in lambs, whilereducing concentrations of trans-11 18:1 (Daniel et al., 2004).


Altering the relative proportions of concentrates and forages in the diet is known tomodify biohydrogenation and formation of fatty acid intermediates in the rumen (Table 12).Experimental data is limited, but it appears that the extent of the changes in the supply offatty acids available for absorption following changes in the F:C ratio are dependent on boththe basal forage, as well as the composition of concentrates. However, it remains unclear asto whether feeding diets with higher proportions of concentrates induces similar changesin ruminal lipid metabolism in grazing cattle or when diets based on grass silage are fed.

Table 12Effect of dietary forage:concentrate ration on the flow of fatty acids at the duodenum in lactating dairy cows

Basal forage Maize silage/Lucerne haylagea Grass hayb

60:40 25:75 Significant 65:35 35:65 Significant

IntakeDry matter (kg/d) 20.6 23.7 P<0.05 20.4 20.5

Fatty acids (g/d)18:1 145 204 P<0.05 56 115 P<0.0518:2 n − 6 379 501 P<0.05 97 142 P<0.0518:3 n − 3 83 58 P<0.05 82 55 P<0.05

Total 789 954 P<0.05 316 395 P<0.05

Flow (g/d)16:0 156 208 P<0.05 72 7418:0 439 535 197 20218:1 cis-9 ND ND 24.0 47.1 P<0.0518:1 cis-11 ND ND 5.20 11.1 P<0.0518:1 cis total 84 140 P<0.05 30.9 66.1 P<0.0518:1 trans-6-8 1.02 3.72 P<0.05 1.83 5.98 P<0.0518:1 trans-9 2.41 4.55 P<0.05 1.38 2.9618:1 trans-10 5.73 29.1 P<0.05 1.46 20.2 P<0.0518:1 trans-11 20.8 33.6 21.4 26.018:1 trans-12 5.68 9.52 P<0.05 1.93 3.7818:1 trans-13 + 14 14.1 22.9 P<0.05 4.17 10.3 P<0.0518:1 trans-15 5.74 8.53 P<0.05 1.95 4.84 P<0.0518:1 trans-16 5.45 7.98 2.34 3.9818:1 trans total 61 120 P<0.05 37.1 80.7 P<0.0518:2 n − 6 86 166 P<0.05 21.8 36.5 P<0.05cis-9, trans-11 CLA 0.33 0.53 0.31 0.31trans-10, cis-12 CLA 0.09 0.26 P<0.05 0.08 0.07trans-9, trans-11 CLA 0.20 0.39 ND NDtrans-10, trans-12 CLA 0.11 0.23 P<0.05 ND NDtrans-11, trans-13 CLA 0.16 0.15 0.33 0.22CLA total 1.07 1.84 2.21 1.7018:3 n − 3 11.2 12.9 8.93 8.82

Total fatty acids 916 1288 P<0.05 427 522 P<0.05

ND, not determined; CLA, conjugated linoleic acid.a Adapted from Kalscheur et al. (1997a) and Piperova et al. (2002).b Data derived from Loor et al. (2004).


Data from recent studies point towards the F:C ratio having more profound effects onruminal biohydrogenation when diets are supplemented with plant oils. Measurements ofduodenal flows of fatty acids in sheep fed rations supplemented with soybean oil and varyingin the F:C ratio from 18:82 to 73:27 (DM basis) indicated that increasing the proportionof Bromegrass hay in the diet was associated with a linear increase in flow of 18:0, cis-9,trans-11 CLA and 18:3 n − 3, and reduced the amounts of cis-9 18:1 and trans-10, cis-12 CLA leaving the rumen (Kucuk et al., 2001). Similarly, increasing the proportion ofBermuda grass hay from 0.12 to 0.36 of total diet DM to steers fed rations containing 20or 40 g sunflower oil/kg DM altered flow of fatty acids from the rumen, irrespective of thelevel of added oil (Sackmann et al., 2003). Changes in ruminal lipid metabolism due to anincrease in diet F:C ratio were characterized as a linear increase in flow of 18:0, trans-11and 12 18:1, cis-11, trans-13 CLA and 18:3 n − 3, and a decrease in trans-10 18:1, trans-10,cis-12 CLA and 18:2 n − 6 at the duodenum (Table 13).

Consistent with results from studies in sheep and steers, recent studies in dairy cowshave also demonstrated the importance of the F:C ratio of the basal ration on ruminal lipidmetabolism when grass hay based diets containing 30 g linseed oil/kg DM are fed (Loor etal., 2004). Under these circumstances, decreases in the F:C ratio enhanced flow of trans-6-8,trans-10, trans-13/14, trans-15 18:1, cis-9 to cis-15 18:1 and 18:2 n − 6 at the duodenum,with a trend to higher amounts of 18:3 n − 3, and reduced amounts of 18:0, escaping therumen (Table 13). Overall, these studies have highlighted that, in addition to the level andtype of lipid supplement, the composition of the basal diet is an important determinant ofthe supply of fatty acids available for absorption and incorporation into milk.

6. Effect of diet composition on milk fatty acid composition responses to lipidsupplements

The final section considers ways in which basal forage type and F:C ratio influence milkfatty acid responses to supplementary lipids. Feeding oils and oilseeds is the most commonnutritional means of manipulating milk fatty acid composition, and it is well establishedthat both the type and source of fat influences the extent of changes that can be achieved(Grummer, 1991; Palmquist et al., 1993; Chilliard et al., 2000, 2001; Lock and Shingfield,2004). Including plant oils in the diet results in a reduction in the short and medium-chainfatty acids, and increases long-chain fatty acids, with responses typically characterized asa shift towards 18:0 at the expense of 16:0 and an overall decrease in the proportion ofSFA and increases in MUFA and PUFA concentrations (Table 14). Reductions in milk fat16:0 concentrations arise from inhibitory effects of long chain fatty acids on mammary denovo fatty acid synthesis, which accounts for approximately 0.5 of the 16:0 secreted in milk(Hawke and Taylor, 1995; Chilliard et al., 2000). Changes in milk fat unsaturated fatty acidsare related to small increases in concentration of the fatty acids predominant in plant oilsupplements, while an increase in milk fat 18:0, cis-9 18:1 and trans 18:1 can be expecteddue to extensive metabolism of long chain PUFA in the rumen which leads to an increasein the amount of 18:0 and trans fatty acids available for absorption (Jensen, 2002).

Examining changes in milk fatty acid composition to diets containing plant oils (Table 14)or fish oil (Table 15) highlights considerable variation in the response when lipid supple-


ments are fed. Part of this can be attributed to differences due to breed of cattle and stageof lactation, as well as the amount of oil included in the ration. However, differences inmilk fatty acid composition responses when oils are fed also arise as a consequence ofthe composition of the basal diet. In an extensive and comprehensive review of the nutri-tional factors affecting the fat composition of caprine milk, both forage type and diet F:Cratio were shown to alter the changes in milk fatty acid composition when supplements

Table 13Effect of dietary forage:concentrate ratio on the flow of fatty acids at the duodenum in steers or cows fed dietscontaining plant oils

Steers (Bermudagrass haya) Dairy cows (Long chop grass hayb)

12:88 24:76 36:64 Significant(LIN)

65:35 35:65 Significant

IntakeDry matter (kg/d) 5.94 6.19 5.54 19.6 20.4

Fatty acids (g/d)18:1 cis-9 75 68 50 P<0.05 139 214 P<0.0518:2 n − 6 170 155 115 P<0.05 181 239 P<0.0518:3 n − 3 15.6 23.0 25.2 P<0.05 445 443

Total 331 329 270 P<0.05 898 1038 P<0.05

Flow (g/d)16:0 52.4 55.8 44.4 97 11418:0 104 152 156 P<0.05 455 31418:1 cis-9 14.3 13.4 12.4 21.6 54.9 P<0.0518:1 cis-11 3.70 4.68 3.87 4.80 18.7 P<0.0518:1 cis-12 0.82 0.54 0.54 3.35 10.5 P<0.0518:1 cis total 18.8 18.6 16.8 35.2 103 P<0.0518:1 trans-6-8 ND ND ND 6.75 16.2 P<0.0518:1 trans-9 0.70 2.28 2.14 P<0.05 3.89 13.118:1 trans-10 41.4 29.8 15.5 P<0.05 6.61 50.6 P<0.0518:1 trans-11 3.48 7.01 11.8 P<0.05 61.7 13918:1 trans-12 1.29 2.49 2.53 P<0.05 8.32 9.5718:1 trans-13 + 14 ND ND ND 29.6 42.9 P<0.0518:1 trans-15 ND ND ND 12.1 16.8 P<0.0518:1 trans-16 ND ND ND 11.5 10.718:1 trans total 46.9 41.6 32.0 P<0.05 145 304 P<0.0518:2 n − 6 13.4 11.9 9.07 P<0.05 20.2 42.8 P<0.05cis-9, trans-11 CLA 0.16 0.21 0.07 0.52 0.86trans-10, cis-12 CLA 0.40 0.26 0.22 P<0.05 0.07 0.10trans-11, trans-13 CLA 0.03 0.01 0.01 P<0.05 1.34 1.37CLA total 0.88 0.79 0.68 3.57 4.7118:3 n − 3 1.08 1.52 1.85 P<0.05 12.9 29.6

Total fatty acids 253 303 283 841 1053 P<0.05a Data reported for steers fed diets of variable forage:concentrate ratio containing 20 or 40 g sunflower oil/kg

diet dry matter (Sackmann et al., 2003).b Data reported for lactating dairy cows fed diets containing 30 g linseed oil/kg diet dry matter (Loor et al.,

2004).

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Table 14Effects of including plant oils in diets with varying basal forage and forage:concentrate ratios on the fatty acid composition of bovine milkLipid source Oil (g/d) Foragea F:Cb Milk fatty acid composition (g/100 g total fatty acids) Reference

4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0 Cis 18:1 Trans 18:1 Total 18:1 18:2 n − 6 18:3 n − 3 CLAControl 0

GS 50:502.9 2.5 1.6 3.7 4.2 12.5 30.1 11.2 19.4 1.6 21.7 1.3 0.40 0.46 Ryhanen et al.

(2005)Rapeseed oil 500 2.6 1.9 1.2 2.5 2.7 10.1 22.6 14.3 25.8 4.3 31.4 1.4 0.50 1.02

Control 0LH 47:53

3.6 2.7 1.7 4.4 5.6 14.8 37.5 6.2 15.1 0.9 16.0 3.6 1.29 ND DePeters et al.(2001)Rapeseed oil 314 3.8 2.7 1.6 4.0 4.8 14.3 30.0 9.6 21.0 1.3 22.3 3.3 1.43 ND

Control 0MS/LH 48:52

5.1 3.7 1.8 5.3 4.7 14.0 32.1 7.9 15.8 1.5 17.3 2.6 0.50 0.50Loor et al. (2002)

Rapeseed oil 815 5.5 3.1 1.3 3.4 3.0 11.3 21.4 11.9 26.4 4.5 30.9 2.5 0.80 1.10

Control 0MS 42:58

1.9 1.9 1.4 3.6 4.4 13.5 33.9 9.5 NR NR 23.2 2.6 0.25 NDJenkins (1998)

Rapeseed oil 618 1.6 1.2 0.8 2.0 2.6 11.0 25.9 13.8 NR NR 35.1 2.1 0.20 ND

Control 0GS 60:40

4.6 2.5 1.3 2.9 3.2 12.4 31.4 15.4 13.0 4.1 17.1 0.9 0.43 0.31 Shingfield et al.(upublished)Soyabean oil 500 4.8 2.2 1.1 2.2 2.4 9.8 24.3 20.0 15.8 7.7 23.5 1.1 0.55 0.53

Control 0GS 60:40

4.0 2.4 1.2 2.8 3.0 11.9 38.2 13.4 11.1 2.4 14.1 0.9 0.41 0.36 Shingfield et al.(upublished)Sunflower oil 500 4.4 2.2 1.1 2.1 2.8 9.3 25.5 23.0 13.8 7.0 21.8 1.3 0.26 0.71

Control 0

MS/LH 60:40

NR NR NR NR NR 13.0 32.2 12.0 22.4 2.9 25.3 4.2 0.80 NRKalscheur et al.(1997b)

High oleicsunflower oil

718 NR NR NR NR NR 8.9 22.0 13.7 28.5 11.8 40.3 4.0 0.61 NR

Sunflower oil 703 NR NR NR NR NR 10.0 23.7 13.8 24.8 11.0 35.8 4.5 0.63 NR

Control 0GH 65:35

3.1 2.4 1.6 3.6 4.1 12.1 29.4 7.1 17.7 2.7 20.4 1.6 0.78 0.62 Loor et al.(2005b)Linseed oil 588 4.0 2.3 1.4 2.5 2.6 9.9 17.2 14.8 31.8 9.0 40.8 1.7 1.19 1.34

Control 0GH 35:65

2.9 2.3 1.5 3.5 3.8 10.2 25.7 6.2 18.3 5.0 23.3 2.2 0.67 0.81 Loor et al.(2005b)Linseed oil 612 2.0 1.1 0.7 1.6 1.9 6.0 18.7 8.1 16.7 12.1 28.8 1.6 1.08 2.54

Control 0GS 60:40

1.8 1.0 0.6 1.8 2.7 9.9 40.2 12.3 19.9 1.1 21.0 2.0 0.72 0.16Offer et al. (1999)

Linseed oil 250 1.8 1.0 0.6 1.5 2.1 8.8 34.0 15.6 25.1 2.1 27.2 1.8 0.84 0.28

Control 0GS 60:40

4.6 2.5 1.3 2.9 3.2 12.4 31.4 15.4 13.0 4.1 17.1 0.9 0.43 0.31 Shingfield et al.(upublished)Linseed oil 500 4.5 2.2 1.1 2.3 2.4 10.0 22.2 20.2 17.3 7.7 25.0 0.7 0.57 0.49

NR, not reported. CLA refers to cis-9, trans-11 conjugated linoleic acid.a GS, grass silage; LH, lucerne hay; MS, maize silage.b Forage:concentrate ratio of the basal diet (DM basis).

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Table 15Effects of including fish oil in diets with varying basal forage and forage:concentrate ratios on the fatty acid composition of bovine milkFish oil source Oil (g/d) Foragea F:Cb Milk fatty acid composition (g/100 g total fatty acids) Reference

4:0 6:0 8:0 10:0 12:0 14:0 16:0 18:0 Cis 18:1 Trans 18:1 18:2 n − 6 18:3 n − 3 CLA 20:5 n − 3 22:6 n − 3Control 0

GS 60:40

1.8 1.0 0.6 1.8 2.7 9.9 40.2 12.3 NR 1.1 2.0 0.72 0.16 0.09 0.04Offer et al. (1999)Tuna orbital 95 1.8 1.0 0.6 1.7 2.5 9.9 39.5 10.5 3.5 1.8 0.71 0.52 0.11 0.07

Fish oil 250 1.8 0.9 0.6 1.6 2.4 10.3 39.6 6.7 9.8 2.5 0.74 1.55 0.11 0.08

Control 0GS 60:40

4.6 2.2 1.1 2.2 2.4 10.2 24.7 19.5 18.1 4.5 0.9 0.42 0.39 0.05 0.00 Shingfield et al.(2003)Herring/mackerel 250 2.4 1.7 1.1 2.8 3.4 13.3 33.3 4.4 4.8 14.4 1.2 0.45 1.66 0.11 0.10

Control 0

LH/MS 50:50

3.2 2.0 1.3 3.1 3.7 11.3 27.1 9.4 16.5 2.4 3.1 0.18 0.60 0.05 0.02Donovan et al.(2000)

Menhaden 290 2.9 1.7 1.0 2.4 3.0 10.4 25.2 7.0 14.5 6.1 2.4 0.36 1.58 0.22 0.06Menhaden 470 2.6 1.4 0.8 1.8 2.3 9.3 26.1 4.4 11.4 12.9 2.0 0.24 2.23 0.32 0.26Menhaden 612 2.9 1.5 0.8 1.9 2.3 9.3 26.6 4.0 10.9 12.1 2.4 0.22 1.90 0.40 0.20

Control 0LH/MS 50:50

3.9 2.5 1.5 3.5 4.0 12.1 29.4 10.4 16.1 1.8 2.6 0.54 0.40 0.05 0.04 AbuGhazaleh etal. (2002)Menhaden 432 3.9 2.3 1.3 2.8 3.2 11.4 27.6 8.1 15.1 3.8 2.2 0.85 0.88 0.24 0.26

Control 0

MS 65:35

4.0 2.3 1.4 3.0 3.3 10.4 27.8 6.5 NR NR 2.0 0.23 0.60 0.05 0.04Chouinard et al.(2001)

Menhaden 184 3.3 1.7 1.0 2.4 2.9 10.9 26.6 3.5 NR NR 1.9 0.23 1.75 0.15 0.54Menhaden 368 3.1 1.6 0.9 2.1 2.6 9.8 24.9 2.5 NR NR 1.6 0.28 1.70 0.35 0.64

Control 0MS 66:34

1.9 1.9 1.3 3.4 4.3 13.9 34.2 8.7 16.7 3.0 1.9 0.28 0.56 0.08 0.04 Loor et al.(2005a)Menhaden 276 1.8 1.8 1.2 3.5 4.5 14.8 31.8 2.7 7.7 13.8 1.4 0.31 3.20 0.36 0.17

Control 0

RG/WC

ND 3.9 1.1 0.9 1.9 2.5 9.5 23.2 12.0 23.6 5.9 2.5 0.99 2.25 0.07 0.06Rego et al. (2005)Sardine 160 ND 3.1 1.0 0.7 1.5 2.0 9.4 23.1 10.4 20.1 8.5 2.0 1.06 3.23 0.18 0.17

Sardine 320 ND 3.1 0.8 0.6 1.4 1.8 8.8 22.6 7.7 15.7 12.0 0.7 1.03 3.64 0.33 0.43NR, not reported; ND, not determined. CLA refers to cis-9, trans-11 conjugated linoleic acid.

a GS, grass silage; LH, Lucerne hay; MS, maize silage; RG/WC, mixed swards of ryegrass and white clover.b Forage:concentrate ratio of the basal diet (DM basis).


of sunflower oil or linseed oil were fed (Chilliard and Ferlay, 2004). Recent research hasshown that the proportion of forage in the diet is also an important determinant of milk fattyacid composition responses in cows fed linseed oil (Table 14), with interactions occurringbetween the F:C ratio of the basal diet and lipid supplementation on the concentration ofseveral fatty acids in milk, including 16:0, 18:0 and cis-9 18:1 (Loor et al., 2005b). Con-sistent with these findings, both the F:C ratio (55:45 DM basis versus 45:55 DM basis)and maize silage particle length (9.52 mm versus 19.05 mm) were found to alter milk fatcomposition from cows fed linseed supplements, with interactions between these factorsbeing significant for milk fat 16:0, cis-9 18:1 and cis-9, trans-11 CLA concentrations (Soitaet al., 2005). Interactions between the level and type of forage in the diet have also beenshown to alter milk fatty acid composition in cows fed a mixture of fish oil and sunfloweroil (Shingfield et al., 2005a). Replacing grass silage with maize silage in the diet resultedin milk fat containing higher amounts of 12:0, 14:0, trans 18:1 and long chain ≥ 20 n − 3PUFA and lower amounts of 18:0 and trans 18:2 (Table 16). Decreases in diet F:C ratiocaused an increase in 18:2 n − 6 and long chain ≥ C20 n − 3 PUFA content, but reducedthe amount of 18:3 n − 3 (Table 16).

Earlier studies provided evidence that changes in ruminal biohydrogenation and milkfatty acid composition occur when relatively low forage diets are supplemented with plantoil PUFA (Griinari et al., 1998; Loor et al., 2004), in response to increased concentratesin the diet (Kucuk et al., 2001; Piperova et al., 2002; Sackmann et al., 2003; Daniel et al.,2004), or by replacing potato starch with a the more rapidly degraded wheat starch in thediet (Jurjanz et al., 2004). These changes are typically characterized by increased formationof trans-10 18:1, and a concomitant reduction in amounts of trans-11 18:1 formed in therumen and corresponding alterations in milk fat 18:1 composition. Because trans-11 18:1can also be converted to cis-9, trans-11 CLA via stearoyl-CoA desaturase activity in themammary gland, and endogenous synthesis is quantitatively the most important source ofthis CLA isomer secreted in milk fat (Griinari et al., 2000; Corl et al., 2001; Piperova et al.,2002; Shingfield et al., 2003; Kay et al., 2004), shifts in biohydrogenation towards trans-1018:1 at the expense of trans-11 18:1 also result in a decrease in milk fat cis-9, trans-11CLA concentrations (Shingfield et al., 2006). The relative amounts of starch and fibre in thediet have often been implicated in inducing changes in the predominant biohydrogenationpathways and formation of specific trans-18:1 isomers (Griinari et al., 1998; Griinari andBauman, 1999; Piperova et al., 2002; Shingfield et al., 2005a), but there is emerging evidencethat milk fatty acid composition responses to lipid supplements are also time dependent.Concentrations of cis-9, trans-11 CLA in milk from diets containing fishmeal and extrudedsoybeans have been reported to increase until 21 days of diet feeding, and decline thereafter(AbuGhazaleh et al., 2004), while levels of this CLA isomer in milk decreased after 14 dayswhen fish oil and extruded soybeans were fed (Whitlock et al., 2002). Similarly, inclusion(45 g/kg diet DM) of a mixture (1:2 w/w) of fish oil and sunflower oil to a maize silage-based ration caused a rapid increase in milk fat cis-9, trans-11 CLA concentrations, whichreached a maximum concentration of 5.37 g/100 g fatty acids within 5 days of diet feeding,but declined thereafter to 2.35 g/100 g fatty acids by day 15 (Shingfield et al., 2006). Earlystudies also demonstrated that milk fat cis-9, trans-11 CLA responses to sunflower oil(Bauman et al., 2000) or soybean oil (Dhiman et al., 2000) were transitory and decreasedwith time. In contrast, inclusion of 50 g/kg of rapeseed oil in concentrates fed to cows


Table 16Effect of forage type and forage:concentrate ratio (F:C) on milk fatty acid composition (g/100 g total fatty acids)from cows fed diets containing (30 g/kg DM) a mixture (2:3 w/w) of fish oil and sunflower oil

Forage Maize silage Grass silage Significant

65:35 35:65 65:35 35:65 F C F×C

4:0 3.35 2.69 3.49 3.176:0 1.62 1.47 1.82 1.618:0 0.85 0.77 0.88 0.8210:0 2.03 1.83 1.78 1.8212:0 2.56 2.39 1.95 2.26 P<0.0514:0 9.68 10.13 8.75 9.00 P<0.0516:0 23.58 25.63 24.18 23.7518:0 4.68 5.49 8.77 7.41 P<0.0518:1 cis-9 10.70 10.30 15.90 13.41 P<0.0518:1 trans-9 0.97 0.96 0.78 0.8418:1 trans-10 7.84 12.73 0.39 7.81 P<0.05 P<0.0518:1 trans-11 8.04 3.48 6.26 4.4818:1 trans-12 1.29 1.01 1.18 1.1118:1 trans-13 and -14 1.36 0.93 1.34 1.2418:1 trans-15 0.49 0.39 0.63 0.58 P<0.0518:1 trans-16 0.33 0.21 0.63 0.44 P<0.0518:1 cis total 12.10 11.83 17.03 14.70 P<0.0518:1 trans total 21.23 20.76 12.01 17.31 P<0.0518:2 trans total 1.13 1.09 1.26 1.41 P<0.05CLA total 3.43 1.91 3.15 2.4318:3 n − 3 0.24 0.20 0.40 0.32 P<0.05 P<0.0520:5 n − 3 0.09 0.09 0.07 0.08 P<0.0522:6 n − 3 0.07 0.06 0.04 0.06 P<0.05 P<0.05

SummaryTotal saturates 52.2 53.6 56.6 54.1Total MUFA 38.5 38.6 34.5 37.0 P<0.05Total PUFA 8.31 7.00 7.82 7.86Total n − 3 PUFA 1.00 0.95 0.91 1.05 P<0.05 P<0.05

Data derived from Shingfield et al. (2005a).

fed grass silage enhanced milk fat cis-9, trans-11 CLA content from 0.46 to 1.02 g/100 gfatty acids, a response that occurred within 7 days of feeding, which was maintained for42 days (Ryhanen et al., 2005). More recently, supplementing relatively high forage dietscomprised of lucerne silage, lucerne hay and barley silage with relatively high levels (60 g/kgdiet DM) of safflower oil or linseed oil reduced milk fat 12:0, 14:0 and 16:0 content andenhanced trans-11 18:1 and cis-9, trans-11 CLA concentrations, responses that were shownto persist during an 8 week experimental period (Bell et al., 2006). Interestingly, inclusionof safflower in combination with 150 mg dl-�-tocopheryl acetate and 24 mg monesin/kgdiet DM was shown to be particularly effective at increasing milk fat cis-9, trans-11 CLAconcentrations from 0.45 to 4.75 g/100 g fatty acid methyl esters (Bell et al., 2006), but theunderlying reasons to explain the sustainable and relatively high milk fat cis-9, trans-11CLA enrichments merits further investigation.


Examining literature data indicates that both the amount and form of the lipid supple-ment, as well as the composition of the basal diet, has a marked effect on changes in milkfat CLA concentrations that can be expected over an extended period of time (Fig. 1).More recent findings also point to the amount and type of forage in the basal diet being animportant determinant. Changes in milk fatty acid composition over an extended period oftime to diets containing relatively high levels of lipid appear to reflect time dependent adap-tations in ruminal biohydrogenation which lead to alterations in the formation of specificintermediates, and alter the supply of fatty acids available for incorporation into milk fat.

The emergence and implementation of legislation on trans fat labelling has resulted inthe oil refining industry investing heavily to reduce the trans fatty acid content of oils andfats used as ingredients for the food processing industry. As a result, the contribution ofindustrial sources to trans fatty acid consumption is declining, such that the remaining transfatty acids in the diet will, for the most part, be derived from ruminant derived foods (Locket al., 2005). Such considerations become increasingly important when lipids such as plantor marine oils are included in ruminant diets to enhance the nutritional quality of milk fatowing to inevitable increase in milk fat trans 18:1 and 18:2 fatty acids. Whilst limited, thereis evidence to suggest that the relative amounts and type of forage in the diet are importantdeterminants of milk fat trans 18:1 (Table 16) and trans 18:2 concentrations (Table 17)when lipid supplements are fed. Unfortunately there is little reliable information to indicate

Fig. 1. Temporal changes in cis-9, trans-11 conjugated linoleic acid (CLA) concentrations (g/100 g fatty acids) inmilk from cows fed diets of different composition containing lipid supplements. [Milk from cows fed diets basedon maize silage (F:C 65:35) containing 45 g/kg DM of a mixture (1:2, w/w) of fish oil and sunflower oil (©;Shingfield et al., 2006), maize silage and hay crop silage (F:C 46:54) containing 52 g sunflower oil/kg DM (�;Bauman et al., 2000), lucerne silage, lucerne hay and barley silage (F:C 60:40) containing either 60 g saffloweroil/kg DM (�; Bell et al., 2006) or 60 g safflower oil + 150 mg dl-�-tocopheryl acetate + 24 ppm monesin/kgDM (+; Bell et al., 2006), lucerne hay and maize silage (F:C 50:50) containing 5 g fish oil and 20 g soyabeanoil/kg DM delivered as fish meal and extruded soyabeans (�; AbuGhazaleh et al., 2004), grass silage (F:C 50:50)supplemented with concentrates containing 50 g/kg of rapeseed oil (�; Ryhanen et al., 2005), or mean responsesof cows fed maize silage (�; F:C 69:31) or grass hay (�; F:C 60:40) based diets supplemented with 50 g sunfloweroil, 50 g linseed oil or 25 g fish oil/kg DM (adapted from Ferlay et al., 2003 and Chilliard and Ferlay, 2004).]


Table 17Effect of forage type and forage:concentrate ratio (F:C) on milk trans 18:2 content (mg/100 g total fatty acids)from cows fed diets containing (30 g/kg DM) a mixture (2:3 w/w) of fish oil and sunflower oil

Forage Maize silage Grass silage Significant

65:35 35:65 65:35 35:65 F C F×C

18:2 cis-9, trans-12 133 119 160 131 P<0.0518:2 cis-9, trans-13 395 324 533 461 P<0.0518:2 trans-9, cis-12 150 113 235 18818:2 trans-11, cis-15 420 440 418 58918:2 trans-9, trans-12 66.4 109.2 33.2 116.818:2 trans, trans 450 489 551 677 P<0.05cis-9, trans-11 CLA 3003 1412 2835 1969cis-11, trans-13 CLA 0.4 0.7 0.8 0.7cis-12, trans-14 CLA 2.0 1.1 4.9 1.4trans-7, cis-9 CLA 168 220 176 180trans-8, cis-10 CLA 38.8 16.1 34.9 20.9trans-9, cis-11 CLA 115 165 26 125 P<0.05trans-10, cis-12 CLA 17.2 26.2 3.1 15.1trans-11, cis-13 CLA 6.4 7.2 14.5 7.7 P<0.05 P<0.05trans-7, trans-9 CLA 14.7 12.1 22.1 24.1trans-8, trans-10 CLA 12.9 15.1 13.5 18.8trans-9, trans-11 CLA 22.3 20.4 27.6 31.0 P<0.05trans-10, trans-12 CLA 9.4 8.9 4.5 9.8trans-11, trans-13 CLA 3.0 3.6 6.0 5.9 P<0.05trans-12, trans-14 CLA 4.9 7.4 9.4 9.2

Data derived from Shingfield et al. (2005a). CLA, conjugated linoleic acid.

the potential impact of these changes on human health related outcomes, but it is perhapsreasonable to be cautious about advocating use of high levels of lipid supplements in thediet to alter milk fatty acid composition until more evidence on isomer specific effects ontrans fatty acids is available.

7. Conclusions

This review has identified a wide range of effects of fresh and conserved forages onmilk fatty acid profiles. The most substantive impacts are those of fresh leafy grass, withwide-ranging improvements in SFA, MUFA and PUFA, and effects of red clover silage inincreasing 18:3 n − 3 in milk fat. It must be acknowledged that even the largest effects offorages are generally much smaller than have been achieved with high levels of concentratefeeding, or the use of ruminally protected (or inert) fats (Dewhurst, 2005). However, use offorages has the attractions of being low-cost with positive consumer perceptions, as well asthat beneficial changes can occur without substantial increases in milk trans fatty acids, agrowing concern with other approaches to manipulate milk fat.

Whilst functional foods are a growth area (Starling, 2002), it has frequently been observedthat consumers expect added-value products without substantial extra cost, suggesting thatlow-cost approaches will be important. In pursuing the low-cost aspect of using forages


to manipulate milk fatty acid proportions, the next steps are to work with plant breed-ers to increase herbage PUFA levels and enhance, and/or optimise, protection and rumenbiohydrogenation mechanisms. Whilst the studies that were reviewed show considerablepotential, there will be enormous challenges associated with interaction effects, such aseffects of growth stage and flowering on differences among genotypes, which typicallycomplicate breeding for plant composition. Some aspects of this research will benefit fromapplication of molecular breeding tools.

Adverse consumer perception of milk fat has been a problem for the dairy industry forsome time and has driven animal breeding and nutrition programs to reduce the fat/proteinratio of milk in many countries. The dairy industry needs to consider whether the growthof low-fat milk and dairy products might be reversed when research increasingly deliverslow-cost health-promoting milk fat. There is ample evidence that consumers appreciatethe sensory experience that fat provides in their diet. Research on milk fatty acids can-not be considered in isolation from consequences for intake, productivity and gross milkcomposition, particularly now that the link between rumen biohydrogenation and milk fatdepression is recognised (Griinari et al., 1998). This research is leading to the point whereit is possible to simultaneously control the level and type of milk fat produced at the farmlevel.

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