Anim. Res. 53 (2004) 213–220
© INRA, EDP Sciences, 2004
Effect of diet on the fatty acid pattern of milk
from dairy cows
Oldemiro A. REGOa*, Paula V. PORTUGALb, Marisa B. SOUSAa,
Henrique J.D. ROSAa, Carlos M. VOUZELAa, Alfredo E.S. BORBAa,
Rui J.B. BESSAb
a Departamento de Ciências Agrárias, Universidade dos Açores, Angra do Heroísmo, Terceira,
b Departamento de Nutrição Animal, Estação Zootécnica Nacional, Instituto Nacional de Investigação
Agrária, Fonte Boa, Santarém, Portugal
(Received 22 April 2003; accepted 24 February 2004)
Abstract – Twelve dairy cows 130 days in milk were sorted by milk production and body weight
and assigned to three feeding regimens in a 3 × 3 Latin-square design, in order to study the effects
of diet on milk fatty acid (FA) composition. The cows were fed a total mixed ration (TMR) consisting
of corn silage (60%) and concentrate (40%) on dry matter basis, or grazed pasture, without (P) or
with 5 kg·d–1 concentrate as a supplement (SP). Supplemented grazing dairy cows produced signif-
icantly more milk than the cows on the TMR and P diets (P < 0.05). The supplementation of grazing
dairy cows with a low fat concentrate did not significantly affect the milk fat FA profile. The pasture
diet, with a supplement or not, decreased the concentration of saturated FA (P < 0.05) and increased
the concentration of unsaturated FA (P < 0.05), of milk fat as compared to the TMR diet. The reduc-
tion in medium-chain FA was offset in large part by increases in long-chain FA (mainly oleic acid).
The concentrations of conjugated linoleic acid (CLA) (P < 0.05) and trans-vaccenic acid were higher
(P < 0.05) in the milk fat from the grazing cows. The results showed substantial variation among
individual cows within treatments on milk fat content of CLA. Significant correlations were found
for individual cow’s milk fat CLA content across diets. Overall, this study indicates that the con-
centration of CLA in milk fat is enhanced by the dietary intake of pasture and that moderate low fat
concentrate supplementation of grazing dairy cows increases performance without compromising
the FA profile of milk fat.
conjugated linoleic acid / dairy cow / fatty acid / milk fat / pasture / total mixed ration
Résumé – Effet du régime alimentaire sur la composition en acides gras de la matière grasse
du lait chez la vache. Douze vaches laitières, en lactation depuis 130 jours, ont été réparties en trois
lots sur la base de la production laitière et du poids vif. Elles ont reçu trois régimes alimentaires,
selon un carré latin 3 × 3, afin d’étudier leurs effets sur la composition en acides gras (AG) du lait.
Les trois régimes étaient une ration complète (TMR) composée d’ensilage de maïs et de concentré
(respectivement 60 % et 40 % sur la base de la matière sèche), de l’herbe pâturée sans
* Corresponding author: firstname.lastname@example.org
O.A. Rego et al.
complémentation (P), ou de l’herbe pâturée complémentée avec 5 kg par jour de concentré (SP). Les
vaches laitières complémentées au pâturage (SP) ont produit significativement plus de lait que celles
qui ne disposaient que du pâturage ou qui recevaient la ration complète. Le pâturage, complémenté
ou non, a diminué la concentration de la somme des AG saturés et a augmenté celle des AG insaturés
(P < 0,05) dans les lipides du lait. La réduction de la concentration des AG à chaîne moyenne a
principalement été compensée par une augmentation de celle des AG à chaîne longue (acide oléique
surtout). La concentration de l’acide linoléique conjugué (CLA) dans les lipides du lait, et celle de
l’acide trans-vaccénique ont été plus élevées (P < 0,05) quand les vaches étaient au pâturage que
quand elles recevaient le TMR. Les résultats ont montré une variation notable entre vaches et intra
régime de la teneur en CLA des lipides du lait, ainsi que des liaisons significatives individuelles entre
les régimes. Notre étude a montré que la teneur en CLA des lipides du lait est accrue par l’ingestion
d’herbe fraîche et qu’une complémentation modérée en concentré augmente la production laitière
sans compromettre le profil en AG.
acide linoléique conjugué / vache laitière / acide gras / pâturage / ration totale mixte
C18:2 cis-9, trans-11, supplied as a natural
constituent of butter manufactured from
In the Azores, the temperate Atlantic cli-
CLA enriched milks, reduces by over 50%
mate presents excellent conditions for the
the incidence of mammary tumours in rats.
implantation of improved pastures allowing
Therefore, the increase in the concentration
milk production to be heavily based on
of this FA in milk can probably exert a ben-
grazing almost all year round. The milk fat
eficial effect on public health.
produced from the pasture has long been
known to be rich in unsaturated fatty acids
The concentration of CLA in cow milk
including trans-octadecenoic acids and
fat can be increased by nutritional manipu-
conjugated isomers of linoleic acid (CLA)
lation, particularly by polyunsaturated lipid
. Fatty acid (FA) composition allied
supplementation . The reason why the
with higher contents of carotene  and its
pasture increases the CLA content of milk
association by consumers with ecological
so clearly is not fully understood. However,
and safe natural products may potentially
it should be considered that pasture contains
increase the health and market value of
high levels of polyunsaturated FA (mainly
these pasture produced milks.
linolenic acid), that certainly contribute as
precursors of CLA and C 18:1 trans-11.
Ruminant milk and fat tissues are the
richest sources of the isomer C
The supplementation of grazing dairy
trans-11, the main CLA isomer, which is
cows with concentrates is a common prac-
produced by rumen bacteria during the bio-
tice and it is recommended when pasture
hydrogenation of linoleic acid  and from
quantity or quality are limiting factors. Feed-
?9 desaturation of trans-vaccenic acid
ing dairy cows on pasture instead of feeding
a total mixed ration (TMR), enhances the
18:1 trans-11) in the tissues of rodents,
ruminants and humans . Trans-vaccenic
concentration of CLA in milk fat . How-
acid is also produced in the rumen during
ever, the information on the effect of a low
the biohydrogenation of linoleic and lino-
fat concentrate, usually used in the supple-
lenic acids . In recent years, the biolog-
mentation of grazing dairy cows on milk FA
ical proprieties of CLA have been under
pattern and particularly on CLA is not well
intensive investigation and it is now well
documented. The objective of this study was,
established that they have potent anticarcino-
therefore, to investigate the effect of feed-
genic effects, modulate immunity, cell dif-
ing regime (TMR vs. pasture with or with-
ferentiation and lipid metabolism . Ip
out concentrate supplementation) on the milk
et al.  demonstrated that the isomer
fat FA profile of dairy cows.
Effect of diet on milk fat composition
2. MATERIALS AND METHODS
Table I. Chemical and FA composition of
concentrates A and B, total mixed ration (TMR)
and pasture (P).
2.1. Animals, feeds and management
The experiment was conducted at the
Conc. AConc. B TMR Pasture
Department of Agriculture Sciences, Uni-
versity of Azores with the European Union
animal welfare directive number 86/609/
EEC. Twelve multiparous Holstein cows in
lactation were blocked based on daily milk
yield (33 ± 7.9 kg), body weight (542 ±
56 kg) and days in milk (130 ± 72 d) and
were randomly assigned to three treatments,
In vitro DMD
in a 3 × 3 Latin-square design. Experimental
Fatty acids (FA)
(g 100·g–1 FA)
periods lasted 22 days during which the
cows were submitted to 3 feeding regimes:
(1) TMR – total mixed ration consisting of
60% corn silage and 40% concentrate A on
a DM basis, (2) P – allowed to graze pasture
alone, (3) SP – allowed to graze pasture sup-
plemented with 5 kg per day of concentrate
B. The ingredients (g·100 g–1 fresh weight)
of the concentrates A and B were respec-
tively 40 and 35 for barley, 5 and 24 for
corn, 43 and 16 for sunflower meal, 0 and
DMD: dry matter digestibility; ADF: acid detergent
20 for corn gluten feed, 7 and 0 for fish meal
fibre; ADL: acid detergent lignin; FA: fatty acid;
and 5 and 5 for a mineral and vitamin premix.
CP: crude protein; CF: crude fat; Conc.: concen-
The collection of samples and data were
made during the last 7 days of each exper-
imental period. In the TMR treatment group,
the animals were fed twice daily in individ-
ual pens with ad libitum access to feed,
according to AOAC  and neutral deter-
allowing 10% orts. The cows on treatments
gent fibre (NDF), acid detergent fibre (ADF)
P and SP grazed as a group (stocking rate
and acid detergent lignin (ADL) according
at 2.5 cows per ha) on a leafy spring ryegrass
to Robertson and Van Soest . In vitro
and white clover pasture. The cows on the
DM digestibility was analysed by the two-
SP treatment received 2.5 kg of concentrate
stage technique of Tilley and Terry . The
B twice a day in the milking room. Pasture
chemical composition, in vitro DM digest-
intake was estimated from animal perform-
ibility and FA composition of all feeds are
ance . The cows were weighed on two
presented in Table I.
consecutive days following a.m. milking at
the end of each period. Twenty plucked
Milk samples were collected from two
samples per hectare of pasture were ran-
consecutive milkings on the last day of each
domly taken daily over the last 7 days of
period and mixed according to milk produc-
each period and mixed in order to obtain a
tion. Milk composition (fat, protein) was
composite sample. These samples were
determined by automated infrared analysis
then stored at –20 °C until assay. Similar
using a Milkoscan 605 (Foss Electric, Hil-
procedures were applied to TMR samples.
lerod, Denmark). Another set of feed and
The samples of feed were analysed for DM,
milk samples were stored at – 20 °C until anal-
crude protein (CP), crude fat (CF) and ash
ysis for FA composition.
O.A. Rego et al.
2.2. Fatty acid analysis
Table II. Dry matter intake, milk production,
milk composition and body weight of cows fed
total mixed ration (TMR), pasture (P) and
Feed samples were weighed into a cul-
concentrate supplemented pasture (SP).
ture tube, and FA methyl esters were pre-
pared by the one-step extraction-methyla-
tion method of Sukhija and Palmquist .
Quantification of FA was done using 4 mg
of 17:0 as the internal standard.
Milk yield (kg·d–1)
25.0a 24.1a 28.5b 0.44
The milk samples were lyophilised and
Milk fat (g·kg–1)
41.7a 41.1a 38.1b 0.40
125 mg of dry solids were then extracted
by chloroform:methanol (2:1) according to
Milk fat yield (kg·d–1) 1.04
Folch et al. . After lipid extraction and
Milk protein (g·kg–1) 32.9ab 32.0b 33.7a 0.21
evaporation of solvents by nitrogen flow in
Milk protein yield
0.82a 0.77a 0.96b 0.01
a 30 °C dry bath, fatty acid methyl esters
(FAME) were prepared by alkaline transes-
Body weight (kg)
terification with methanolic KOH . FAME
On each line, the values with different letters are
were analysed by gas chromatography (GC),
significantly different (P < 0.05).
using a 60-m fused silica capillary column
DMI: dry matter intake.
SP-2380 (Supelco, Bellefonte, PA, USA)
with 0.25-mm internal diameter and 0.20-µm
film thickness. A HP5890A series II chro-
2.3. Statistical analyses
matograph (Hewlett-Packard, Avondale, PA,
Data were analysed as a replicated 3 × 3
USA) working with nitrogen as the carrier
Latin-square using the GLM procedure of
gas and a flame ionisation detector was used.
SAS  with the following model: Y =
The initial column temperature of 130 °C
µ + TI + Pj + Ak + ?ijk, where Yijk is the
was held for 10 min, increased to 165 °C at
dependent variable, µ is the global mean,
5 °C per min and held for 5 min. Then, the
T is the treatment effect, P is the period
temperature was increased to 180 °C at 3 °C
effect, A is the animal effect and ?ijl is the
per min and held for 6 min. Then the tem-
residual error. The values presented are
perature was increased at a rate of 1.5 °C per
least square means followed by standard
min until 230 °C, where it remained for
error of mean (SEM).
25 minutes. The injector and detector tem-
peratures were 230 °C and 280 °C, respec-
tively. Peak identification was based on co-
3. RESULTS AND DISCUSSION
chromatography with known standards of
FAME (Sigma, St. Louis, USA). The trans-
Concentrates A and B had very similar
C18:1 isomers are reported as one value, since
FA profiles, despite the differences in for-
this column incompletely resolves them,
mulation and chemical composition (Tab. I).
and we cannot exclude some minor contam-
The in vitro DM digestibility of pasture was
ination with other 18:1 isomers. The CLA
5% higher than the TMR diet.
was computed as the major peak in the con-
Data of DM intake, milk production,
jugated octadecadienoic region of the chro-
milk composition, and body weight of cows
matogram that had an elution time consist-
have been previously presented and dis-
ent with cis-9,trans-11 octadecadienoic
cussed by Sousa et al.  and can be seen
FAME (Sigma, St. Louis, USA). FA were
in Table II. Dry matter intake did not differ
expressed as g·100 g–1 of reported fatty
significantly between treatments. The esti-
mated intake of metabolisable energy (ME)
Effect of diet on milk fat composition
was 220, 206 and 232 MJ per day, respec-
Table III. Fatty acid (g 100·g–1 FA) of milk fat
tively, for TMR, P and SP cows, which can
from cows fed total mixed ration (TMR), pasture
explain the differences in animal perform-
(P) and concentrate supplemented pasture (SP).
ance. Supplemented grazing dairy cows
produced significantly (P < 0.05) more milk
(16% with a response of 0.88 kg milk·kg–1
concentrate) with lower fat (resulting in no
effect on fat yield) and higher protein con-
tents. These results are consistent with the
greater nutritive value (more ME·kg DM–1)
of grazed pasture relative to the TMR diet
(Tab. I), but contrast with the results of
Kolver and Muller .
The effect of the treatments on the FA
profile of milk fat is present in Table III. The
concentrate supplementation of grazing dairy
cows (SP) did not exert a significant effect
cis9- C18:1 18.7a
on the composition of principal FA of milk
fat, including CLA and C 18:1 trans-11, rel-
ative to the P treatment (Tab. III). This can
be explained by the fact that the low fat con-
centrate (Tab. I) supplemented to pasture in
the SP treatment group represented only a
small fraction (about 12%) of total lipid
intake. Probably this amount of supple-
UFA:SFA ratio 0.37a
mented lipids, mainly as linoleic acid, is not
enough to modify the rumen environment
and FA biohydrogenation pattern. Pasture
based diets (P and SP) decreased by 32% the
On each line, the values with different letters are
amount of hypercholesterolemic saturated
significantly different (P < 0.05).
FA on milk fat (sum of C
CLA: cis9,trans11-C18:2 isomer;
12:0, C 14:0 and
UFA: sum of unsaturated fatty acids (C
16:0) and increased the amount of monoun-
C18:1, trans-C18:1, C18:2, CLA, C18:3);
saturated (sum of C 16:1 cis-9, C 18:1 trans-
SFA: sum of saturated fatty acids (C10:0, C12:0,
11 and C
18:1 cis-9) and polyunsaturated FA
14:0, C15:0, C16:0, C17:0, C18:0);
(sum of C
MUFA: sum of monunsaturated FA (C
18:2 n-6, CLA and C 18:3) respec-
tively by 33 and 43%, relative to the TMR
PUFA: sum of polyunsaturated FA (C
diet (P < 0.05). The intake of pasture
increased (P < 0.05) the linolenic acid con-
tent of milk fat almost 2.5 fold, in agreement
with Kelly et al.  and Dhiman et al. .
Milk fat from cows grazing pasture (sup-
Increases in concentrations of C 18:0 and
plemented or not) had 2.5 times more CLA
C 18:1 trans-11 and C 18:1 cis-9 in milk fat
cis-9, trans-11 isomer than milk fat from the
and decreases in C 16:0 are typically observed
TMR diet (Tab. III). The effects of intake
in grazing cows, when compared to those
of pasture on CLA concentration in milk fat
fed TMR diets or conserved forages .
are now well established [4, 8]. Kelly et al.
Rego and Almeida  observed that replac-
 reported that a diet exclusively based
ing pasture by maize and grass silage
on pasture increases the levels of CLA in
induces an increase of short and medium-
milk fat by 2.5 fold relative to TMR. In
chain FA in milk fat and a decrease of long-
agreement with this, Dhiman et al.  found
chain FA, particularly of oleic acid.
a linear increase in CLA levels in milk fat
O.A. Rego et al.
ments were pooled together. The slope of
the regression line obtained from TMR data
was different from that obtained for P and
for SP. The close relationship between
CLA/C18:1 trans-11 has been reported by
several authors, using a great spectrum of
diets, as reviewed by Chilliard et al. .
Despite the limitation of the experimen-
tal design, a high variation in milk fat CLA
content can be inferred for cows within each
treatment. The variation limits were, 0.21 to
1.52, 0.31 to 2.38 and 0.43 to 2.75 g·100 g–1
Figure 1. Relationships between the concentra-
respectively for TMR, P and SP diets. Sev-
tions of C18:1 trans-11 and CLA in milk fat
eral studies [12, 14, 24], report a wide var-
from cows fed either TMR (o) or P (•) and
iation of CLA levels of milk fat among
Regression equations: TMR: y = 0.22 ± 0.017
cows. The correlations between the milk fat
x + 0.05 ± 0.044, n = 12, r2 = 0.94, P < 0.0001;
CLA contents for individual cows across
P and SP: y = 0.41 ± 0.052 x – 0.32 ± 0.214,
diets are shown in Table IV. The correla-
n = 24, r2 = 0.74, P < 0.0001.
tions were statistically significant for the
TMR × P and TMR × SP diets and close to
significance for the P × SP diets. Lawless
et al.  showed significant correlations
when pasture is increased in the diet of dairy
between levels of CLA in milk fat from indi-
cows. A survey study of CLA content in the
vidual cows within the same breed, in two
milk fat of ruminants, involving several
distinct samplings, on pasture. On the con-
farms, showed a typical seasonal variation,
trary, Solomon et al.  observed that indi-
with a maximum concentration in the spring-
vidual cows did not maintain the same rel-
summer, which coincides with the grazing
ative rank in milk CLA concentration across
period, and a minimum concentration in the
diets. However, Peterson et al.  reported
winter when animals are kept indoors .
that the individual hierarchy for milk fat
The reason why pasture grazing increases
CLA content was maintained to a large
milk CLA so clearly is not fully understood.
extent over a 12-week study even in the var-
Fresh grass contains 1 to 3% FA, of which
iable treatment group that alternated between
55 to 65% is ?-linolenic acid . Linolenic
two diets. The physiological basis for the
acid pathways in the rumen are not known
variation between individuals in CLA lev-
to involve CLA as an intermediate, although
els of milk fat and the individual rank kept
C18:1 trans-11 is produced . This FA is
a known precursor of endogenous synthesis
Table IV. Correlation coefficients between the
18:2 cis-9, trans-11 in the lactating
mammary gland . Other specific factors
CLA contents of milk fat of the individual cows
affecting the rumen microbial ecology may
across diets (n = 36).
contribute to CLA production when cows
are switched to pasture.
A close relationship between concentra-
tions of CLA (cis-9, trans-11) isomer and
(P < 0.09)
(P < 0.02)
18:1 trans-11 in milk fat was observed in
this study (Fig. 1). Slopes of the regression
(P < 0.01)
lines for P and SP treatments did not differ
SP: concentrate supplemented pasture, TMR:
statistically and the data from both treat-
total mixed ration, P: pasture.
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