ETH Library
A study on the causes for the
elevated n−3 fatty acids in cows'
milk of alpine origin
Journal Article
Author(s):
Leiber, Florian; Kreuzer, Michael; Nigg, Daniel; Wettstein, Hans-Rudolf; Scheeder, Martin Richard Leo
Publication date:
2005-02
Permanent link:
https://doi.org/10.3929/ethz-b-000033135
Rights / license:
In Copyright - Non-Commercial Use Permitted
Originally published in:
Lipids 40(2), https://doi.org/10.1007/s11745-005-1375-3
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ABSTRACT: The influence of grass-only diets either from rye-
grass-dominated lowland pastures (400 m above sea level) or
botanically diverse alpine pastures (2000 m) on the FA profile of
milk was investigated using three groups of six Brown Swiss cows
each. Two groups were fed grass-only on pasture (P) or freshly
harvested in barn (B), both for two experimental periods in the
lowlands and, consecutively, two periods on the alp. Group C
served as the control, receiving a silage-concentrate diet and per-
manently staying in the lowlands. Effects of vegetation stage or
pasture vs. barn feeding on milk fat composition were negligible.
Compared with the control,
α-linolenic acid (18:3n-3) consump-
tion was elevated in groups P and B (79%;
P < 0.001) during the
lowland periods but decreased on the alp to the level of C owing
to feed intake depression and lower 18:3n-3 concentration in the
alpine forage. Average 18:3n-3 contents of milk fat were higher
in groups P and B than in C by 33% (
P < 0.01) at low and by 96%
(
P < 0.001) at high altitude, indicating that 18:3n-3 levels in milk
were to some extent independent of 18:3n-3 consumption. The
cis-9,trans-11 CLA content in milk of grass-fed cows was higher
compared with C but lower for the alpine vs. lowland periods
whereas the
trans-11,cis-13 isomer further increased with alti-
tude. Long-chain n-3 FA and phytanic acid increased while ara-
chidonic acid decreased with grass-only feeding, but none of
them responded to altitude. Grass-only feeding increased milk
α-
tocopherol concentration by 86 and 134% at low and high alti-
tude (
P < 0.001), respectively. Changes in the ruminal ecosystem
due to energy shortage or specific secondary plant metabolites
are discussed as possible causes for the high 18:3n-3 concentra-
tions in alpine milk.
Paper no. L9566 in Lipids 40, 191–202 (February 2005).
Recently
, elevated contents of beneficial functional F
A in
cows’ milk and cheese derived from alpine grazing systems
were reported (1–4). Increased contents of n-3 PUFA and an
improved ratio of n-3 to n-6 PUF
A in milk of cows grazing on
alpine pastures were shown (3,5). It was even hypothesized that
a relation between high levels of
α-linolenic acid (18:3n-3) in
alpine cheese and a favorable cardiovascular health status of
the alpine population may exist (4). Generally, n-3 PUFA are
known to be essential for human health, and problems may
arise if they are consumed in too low a proportion relative to
n-6 FA (6). Furthermore, the content of CLA (18:2
cis-9,trans-
11 isomer), a FA that also has shown potential to benefit human
health (7), was found to be clearly enriched in milk (3) or
cheese (1,2) from alpine production systems compared with
those from intensive lowland production. Additionally, the pro-
portion of short- and medium-chain saturated FA (SFA), which
are supposed to increase the risk of cardiovascular diseases (8),
was found to be markedly lower in milk and milk products
originating from cows grazing high alpine pastures (2,4,5).
Milk fat derived from cows grazing high alpine pastures
therefore seems to have a considerably higher dietetic value
than conventional milk, but the reasons for these differences
are still unclear. To our knowledge, in previous studies the in-
dividual factors of the alpine sojourn of cows potentially af-
fecting milk fat composition were never differentiated, and
diets fed in the lowlands for control were either not specified
or not restricted to herbage-only, as is usual practice in Swiss
alpine dairy systems. Known mechanisms influencing the FA
profile in lowland milk are (i) the basic effect of grazing and
the related body fat mobilization (9,10) as well as influences of
the herbage’s botanical composition (10,11), (ii) the degree of
usage of concentrates, in particular wheat, barley
, and maize in
the diets, which mainly contribute SFA, 18:1, and 18:2n-6 but
very little 18:3n-3 (10,12,13), and (iii) the effect of herbage
conservation (13–15). These factors could also contribute to
the differences in composition between common lowland milk
and milk from alpine pastures. It is, however, still unclear
whether
, in addition, specifi
c plants (16), the 18:3n-3 content
of the alpine flora (3,17) and even the hypoxic environment
per
se
(4) are responsible for the typically high contents of CLA
and n-3 PUFA of alpine milk products. Otherwise, the “alpine
paradox” (4) would actually be a general phenomenon of feed-
ing grass at any altitude and mainly reflect the difference from
mixed, often maize-based, diet types. The objective of the pre
-
sent study was to determine experimentally the factors respon-
sible for the composition of milk fat of cows consuming grass
at high altitude. Comparisons included grass-only vs. mixed
diets in the lowland, high altitude vs. lowland grazing of the
cows, pasture vs. indoor feeding, and young vs. mature swards.
Copyright © 2005 by AOCS Press 191 Lipids, Vol. 40, no. 2 (2005)
*To whom correspondence should be addressed at Institute of Animal Sci-
ence, ETH Zurich, ETH-Zentrum/LFW B56, CH-8092 Zurich, Switzerland.
E-mail: michael.kreuzer@inw.agrl.ethz.ch
Abbreviations: 18:3n-3, α-linolenic acid; B, barn group; BHB, β-hydroxy-
butyrate; C, control group; CRC, controlled release alkane capsules; DM,
dry matter; LS, least squares; NEFA, nonesterified FA; P, pasture group;
SFA, saturated FA.
A Study on the Causes for the Elevated n-3 Fatty Acids
in Cows’ Milk of Alpine Origin
Florian Leiber, Michael Kreuzer*, Daniel Nigg,
Hans-Rudolf Wettstein, and Martin Richard Leo Scheeder
Institute of Animal Science, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland
EXPERIMENTAL PROCEDURES
Animals. Eighteen Brown Swiss cows, having been 94 (65 to
127) d into their second lactation at the beginning of the exper-
iment, were blocked by calving date and allocated to three
groups (P, pasture group; B, barn-fed group; C, control group)
with minimal between-group variance for energy-corrected
milk yield, milk protein, and milk fat percentage. Average milk
yield, at the start of the experiment, was 21.7, 22.2, and 22.3
kg/d for groups P, B, and C, respectively. The corresponding
average contents per kilogram of milk were 43.6, 42.0, and
41.4 g fat; 30.3, 31.1, and 31.8 g protein; and 50.6, 50.4, and
49.1 g lactose, respectively.
Experimental schedule. Six subperiods (S0 to S5) were de-
fined. Each subperiod lasted for 21 d, and intensive sampling
was always done during the respective third week. All data and
data points in the figures refer to the average values of these in-
tensive sampling weeks within each subperiod. The first part
of the experiment took place at the ETH research station
Chamau in central Switzerland at 400 m above sea level. Dur-
ing the baseline subperiod (S0) all cows were tethered in a barn
and fed a mixed control diet. During the “lowland period,”
comprising the two subperiods S1 and S2, group P was turned
out to pasture for 24 h/d, while groups B and C remained in the
barn. Group B was fed only freshly harvested grass
ad libitum,
and group C remained on the control diet. After S2, all cows
were kept in the barn again until the alpine vegetation was
ready for grazing and harvest. During this time, groups P and
B stayed on the grass-only diet at ad libitum access. At the end
of this transition period, which lasted for 28 d, just before and
after the transport to the alpine location, additional milk sam-
ples were taken. These samples provided the additional data
points in Figures 3 to 6. For the “alpine period,” Groups P and
B were transported to the alpine ETH research station Alp
Weissenstein located at 2000 m above sea level in the Eastern
Swiss alps. Half of the group P and B cows were switched to
the respective other group in order to counterbalance possible
residual effects of the previous treatment. This balanced re-
arrangement of groups P and B is indicated in the figures by
the dotted line. Group C remained in the lowlands and contin-
ued to serve as control group. Like the lowland period, the
alpine period consisted of two subperiods (S3 and S4). S5 as a
final baseline evaluation was carried out with all cows kept in
the lowland barn again and fed the control diet.
During the intensive sampling weeks, milk yield was
recorded and proportionate milk samples of each cow and each
milking were obtained. The samples were frozen at
−20°C immediately after sampling. Cows were weighed and
blood samples were taken at days 2 and 6 within each sampling
week. Two feed samples were drawn every day in each group
during the sampling weeks of the subperiods. An aliquot of
each feed sample was dried at 60°C for 48 h, and a second was
frozen at −20°. Feces were sampled once a day from each ani-
mal and frozen. Later
, all samples were defrosted, pooled per
sampling week (feed) or per animal and sampling week (feces),
and then dried and ground through a 0.75-mm sieve for analy-
sis. Feed intake was measured daily for cows kept in barn. On
pasture, feed samples were obtained mimicking the selection
behavior of the cows for at least 3 h distributed over the whole
daytime, and feed intake was calculated using the double-
alkane technique (18). As the source for the synthetic marker
alkanes (C
32
and C
36
), controlled release alkane capsules
(CRC; Captec, Auckland, New Zealand) were introduced into
the rumen of the cows 8 d prior to each sampling week. Odd-
chain alkanes (C
31
and C
33
) in feed were used as internal mark-
ers. The actual alkane recovery rates in feces were determined
with the known feed intake of the group B cows that had also
received CRC.
Diets. S1 started at the earliest possible vegetation stage of
the first growth; S2 followed 3 wk later on a now more mature
sward. The same principle was followed for S3 and S4 at the
alpine location. The lowland areas were intensively managed
leys with regular manure fertilization. The alpine pasture was
native and not fertilized. The botanical composition was visu-
ally estimated in the standing sward within each sampling week
in eight systematically distributed 9-m
2
plots (four per pasture
and harvest paddock, respectively) according to the method of
Braun-Blanquet (19). The main species characteristics of the
experimental sites are shown in Table 1. The analyzed nutrient
and FA composition of the experimental feeds is listed in Table
2. Data for individual vegetation stages are not displayed since
vegetation stage effects on milk fat composition were found to
be not significant.
Herbage for group B was harvested daily in the early morn-
ing from paddocks adjoining those grazed by group P cows;
the two were similar in botanical composition. It was then kept
tightly packed to avoid any possible losses of FA by wilting of
the forage (15). The control diet contained hay, ryegrass silage,
and maize silage in proportions of 0.1:0.6:0.3 on a dry-matter
(DM) basis, was provided
ad libitum, and was supplemented
by concentrates according to milk yield. The two commercial
concentrates used contained rumen-protected fat (Table 2),
which made up 410 g/kg of total dietary FA in the control diet.
The cows of all groups had
ad libitum access to NaCl and a
nonvitaminized mineral mix (Ca:P = 2:1; Nährkosan, Büron,
Switzerland) during the whole experiment.
Analysis. Frozen milk samples were defrosted and pooled
per cow and sampling week. After direct transesterification, ac-
cording to Suter et al. (20), the FAME were extracted with n-
heptane and then separated and quantified with GC using a Sil
88 column (100 m
× 0.25 mm, 0.2 µm; Varian Inc, Darmstadt,
Germany) on an HP 6890 (Hewlett-Packard, Wilmington, DE).
Hydrogen fl
ow was 1.5 m
L/min; 0.5 mL of
n-heptane contain
-
ing the FAME was injected on column. The temperature pro-
gram was: 60°C for 5 min; increase by 14°C/min up to 165°C;
2°
C/min up to 225°C; isothermal for another 15 min. Tempera-
ture program and peak identification were adapted from Col-
lomb and Bühler (21) with some modifications. The peaks for
16:1
trans and 20:1trans may contain some branched-chain
FA
(22). They were therefore only included in the calculation of
total FAME and not separately shown in the tables. The applied
chromatographic method also is not able to separate
trans-
192 F. LEIBER ET AL.
Lipids, Vol. 40, no. 2 (2005)
8,cis-10 and trans-7,cis-9 from the major cis-9,trans-11 CLA-
isomer as well as trans-11,cis-13 from cis-9,cis-11 (3,23).
However, the concentration of the
trans-7,cis-9 isomer has
been described to be about 100-fold lower than the cis-9,trans-
11 isomer in milk, and changes due to (alpine) feeding occurred
on a rather low level (3,24). Proportions of and effects on trans-
8,cis-10 are even lower than for trans-7,cis-9 (24). Thus, we
conclude that any distortion of the effect on the
cis-9,trans-1
1
isomer is small, although it might be slightly biased because
both of these minor isomers are likely to increase with altitude
of the site where the dairy cows are kept (24). Similarly
, the
cis-9,cis-11 isomer, coeluting with the trans-11,cis-13 isomer,
seems to be present only in traces if at all (24; Collomb, M.,
personal communication); we therefore refer to this peak as
trans-11,cis-13. Response factors were determined using BCR
164 standard milk fat (Certified Reference Material; EC Refer-
ence Materials, Brussels, Belgium) as a reference. The milk fat
content measured with a standard NIR procedure (Milkoscan
4000; Foss, Höganäs, Sweden) closely correlated with the
amount of total FAME obtained with GC (r
2
= 0.93). Phytanic
acid (3,7,11,15-tetramethylhexadecanoic acid) was separated
and quantified in the milk samples of S2 and S3 and in the sam-
ples drawn directly before and after transfer to the alp, using an
RTX225 column (30 m × 0.32 mm, 0.5 µm; Restek Corpora-
tion, Bellefonte, PA). Injection volume was 1 µL in a 30:1 split
modus at 240°C. Hydrogen flow was 4.0 mL/min. The temper-
ature program was 140°
C for 2 min, ramp 5°C/min up to
215°C, isothermal for 5 min.
Lipids from feeds were extracted by accelerated solvent ex-
traction (ASE 200; Dionex Corp., Sunnyvale CA) using
hexane/isopropanol (2:1 vol/vol) and transformed into FAME
according to Wettstein et al. (25). FAME were dissolved in
hexane and analyzed with GC on a Supelcowax
™
-10 column
(30 m
× 0.32 mm, 0.25 µm; Supelco Inc., Bellefonte, PA) after
split injection (1:30) at 270°C. The injection volume was 2 µL;
hydrogen flow was 2.2 m
L
/min. The temperature program was
160°C for 1 min, ramp 20°C/min up to 190°C, ramp 7°C/min
up to 230°C; isotherm at 230°C for 4 min, ramp 20°C/min up
to 250°
C, isotherm for 4 min.
Tocopherols in milk were analyzed after saponification with
KOH with normal-phase HPLC on a Merck-Hitachi system
with UV detection (La Chrom; Darmstadt, Germany) accord
-
ing to Rettenmaier and Schüepp (26). The
β-, γ-, and δ-tocoph-
erols were always below the detection limit.
Feed contents of DM and nitrogen (N × 6.25 = crude pro
-
tein) were analyzed with standard methods as described previ-
N
-3 FATTY ACIDS IN ALPINE MILK 193
Lipids, Vol. 40, no. 2 (2005)
TABLE 1
Frequent Plant Species in the Lowland and the Alpine Pastures Used in the Experiment
Estimated proportions of total ground covering
a
>10% 4–10% 1–4%
Lowland site (16 plots)
Monocotyledonae (77%)
Total species found: 9
Lolium multiflorum, Poa trivialis, Poa annua Festuca pratense, Poa pratense
Lolium perenne
Leguminosae (6%)
Total species found: 2 Trifolium repens Trifolium pratense
Dicotyledonae (except
Leguminosae) (16%)
Total species found: 8 Taraxacum officinale Rumex obtusifolius,
Ranunculus repens, Bellis
perennis
Alpine site (16 plots)
Monocotyledonae (36%)
Total species found: 14 Festuca rubra Trisetum flavescens, Phleum Poa trivialis, Nardus
alpinum, Poa alpina, Festuca stricta, Poa pratense
pratense
Leguminosae (23%)
Total species found: 11
Trifolium pratense Trifolium montanum Trifolium repens, Trifolium
badii, Lotus
spp.,
Trifolium nivale
Dicotyledonae (except
Leguminosae) (40%)
Total species found: 46 Rumex arifolius, Alchemilla Peucedaneum osthrutium,
vulgaris, Taraxacum officinale Ranunculus montanus,
Ranunculus acer, Achillea
stricta, Carum carvi, Chaero
phyllum cicutaria, Campanula
scheuchzeri, Crepis aurea,
Veronica chamaedrys,
Plantago alpina,
Leontodon hispidus
a
Visually estimated according to the method of Braun-Blanquet (19).
ously (27). Contents of net energy for lactation were calculated
based on the equations used in the official Swiss feed evalua-
tion (28). Plasma metabolites and insulin were analyzed with
standard assays. Plasma insulin was measured with a radioim-
munoassay kit (No. 1064 1401; Pharmacia, Uppsala, Sweden).
Plasma concentrations of nonesterified FA (NEFA), β-hydrox-
ybutyrate (BHB), glucose, and urea were analyzed on a
COBAS-MIRA chemistry analyzer (Roche, Basel, Switzer-
land), with the following assays: NEFA with NEFA-C WA
994-75409 (Wako, Neuss, Germany), BHB with product 310-
A (Sigma, Buchs, Switzerland), glucose with assay No.
1447513 and urea with assay No. 1489364 (both Roche).
Statistics. Data were evaluated with three mixed models by
SAS V8.2e (PROC MIXED; SAS Institute, Cary, NC;). Model
1 included group (P, B, or C) and calving date (classified in
three blocks of similar calving date) as fixed factors. Animal
was included as random effect, and the individual baseline (S0)
values were used as a covariate. The least squares (LS) means
and all significances of the group
× period interaction, shown
in Tables 3 and 4, are based on model 1. The same model, but
without the covariate S0, was used to obtain LS means of all
subperiods (including S0) for the illustration in the figures. A
third model was applied for the calculation of the altitude and
the vegetation stage effects, considering vegetation stage (two
classes: young or mature) and main period (lowland or alps) as
additional fixed effects. Group C, for which feed and altitude
remained unchanged, was excluded from the calculations with
model 3.
RESULTS
Feed intake and metabolic energy supply. During the lowland
period, the DM intake of the grass-only fed cows was lower
than in cows of group C [significantly for group P (
P < 0.05)
and as a tendency for group B (P < 0.1); Table 3]. Nevertheless,
the calculated net energy intake of B and P cows did not differ
significantly from C cows during the lowland period (Fig. 1A),
and milk yield decreased only slightly (Fig. 2B). However, un-
like C cows, P and B cows lost weight from S0 to S1 (
P < 0.05;
Fig. 2A), and all blood plasma metabolites indicated a certain
catabolic state (Table 3). In the alpine period (S3 and S4), DM
intake in groups P and B decreased (
P < 0.001) compared with
the lowland period and with C cows, and net energy intake was
reduced by approximately one-third (
P < 0.01). Accordingly,
milk yield markedly decreased when cows were transferred to
the alpine location (
P < 0.001; Fig. 2). Although body weight
did not further decline, plasma levels of BHB (P < 0.001) and
NEFA (P <
0.001) still increased and plasma glucose (P <
0.05) and insulin (P < 0.001) further decreased during the
alpine sojourn of the cows (Table 3).
FA intake. In S1 and S2, individual FA consumption of the
grass-only fed cows differed from that of the C cows (Table 3)
since lowland grass had clearly higher levels of 18:3n-3 but
much lower levels of SFA, monounsaturated FA, and 18:2n-6
than the control diet (
cf. Table 2). Thus, 18:3n-3 was the main
dietary F
A for groups P and B, whereas for group C, 16:0 and
194 F. LEIBER ET AL.
Lipids, Vol. 40, no. 2 (2005)
TABLE 2
Nutrient and FA Contents of Feeds Used in the Experim
ent
a
FA (g/kg DM)
Dry matter (DM) NE
L
b
Crude protein Total
Origin
(g/kg) (MJ/kg) (g/kg)
FA
SFA
c
18:1
∆11
18:1
∆9
18:2n-6 18:3n-3
Green forages
d
Pasture
Lowland
190
± 13 6.12
± 0.46 142
± 4
16.2
± 3.7 2.86
± 0.60
0.065
± 0.029 0.470
± 0.288
2.91
± 1.16 9.06
± 1.61
Harvest
Lowland
177 ±
24 6.11
± 0.54 137
± 8
14.8 ±
2.7 2.77
± 0.47
0.057 ± 0.019 0.343
± 0.177
2.61 ±
1.01 8.27
± 0.87
Pasture
Alpine
190 ± 4
5.57 ±
0.41 138
± 8
12.2 ± 2.4 2.77
±
0.33 0.066
± 0.012 0.616
± 0.037
2.35 ± 0.42 5.72
± 1.45
Harvest
Alpine
186 ±
25 5.63
± 0.28 138
± 8
13.4 ±
2.4 2.93
± 0.37
0.069 ± 0.004 0.646
± 0.004
2.63 ±
0.34 6.29
± 1.50
Components of the
mixed diet
d
Hay
Lowland
770
± 14 5.62
± 0.02 178
± 1
18.4
± 0.1 4.64
± 0.09
0.085
± 0.000 0.439
± 0.003
2.90
± 0.03 9.12
± 0.19
Grass silage
Lowland
514 ± 43 5.32
± 0.03 168
± 0 19.0
± 0.3 4.40
± 0.19
0.080
± 0.006 0.669
±
0.043
3.19 ± 0.11 9.58
±
0.33
Maize silage
Lowland
334 ± 11 6.33
±
0.01 62
± 0
22.8 ± 0.2 4.57
±
0.06
0.205 ± 0.004 6.103
± 0.177 11.20
± 0.20 0.59
± 0.01
Energy concentrate
e
Lowland
854
± 2 7.45
± 0.05 151
± 4
61.3 ±
1.6 44.79
± 1.54
0.335 ±
0.016 5.587
± 0.324
9.70
± 0.70 0.78
± 0.02
Protein concentrate
f
Lowland
860
± 0 7.75
± 0.05 423
± 6
59.1 ±
0.6 46.23
± 0.55
0.275 ±
0.006 4.229
± 0.366
7.68
± 0.47 0.57
± 0.09
a
Means
± SD.
b
NE
L
: net energy for lactation, calculated based on formu
la of RAP (28)
c
SFA: saturated FA (sum of 10:0 through 24:0).
d
Average of two subperiods with two samples per sub
period; each sample pooled from 7 d.
e
g/kg DM: wheat, 300; barley, 190; wheat bran, 150
; oats, 120; straw meal, 50; maize, 50; crystalline fat (rum
en-protected; Alikon, Bützberg, Switzerland), 40; maize g
luten, 30; molasses, 30; canola cake, 20;
sugar beet pulp, 20.
f
g/kg DM: soybean meal, 480; maize gluten, 220; str
aw meal, 60; maize, 50; wheat, 40; crystalline fat, 40; su
gar beet pulp, 40; molasses, 30; wheat bran, 10.
