Comm. Appl. Biol. Sci, Ghent University, 73/1, 2008
1
EFFECT AND WORKING MECHANISM OF DHA ON
RUMEN BIOHYDROGENATION OF LINOLEIC ACID AND
IMPLICATIONS FOR MILK FAT OPTIMISATION
1
C. BOECKAERT1,2, N. BOON2, W. VERSTRAETE2 & V. FIEVEZ1
Laboratory for Animal Nutrition and Animal Product Quality, Ghent University,
Proefhoevestraat 10, 9090 Melle, Belgium
2
Laboratory for Microbial Ecology and Technology, Ghent University, Coupure Links 653, 9000
Gent, Belgium
INTRODUCTION
Milk is a source of conjugated linoleic acids of which CLA c9t11 has anticarcinogenic and anti-atherogenic effects (Jensen, 2002). CLA in milk originates from biohydrogenation of dietary lipids in the rumen. On the one
hand, CLA is formed directly during the microbial hydrogenation of linoleic
acid (C18:2 n-6) in the rumen. On the other hand, CLA is derived from the
endogenous conversion of vaccenic acid (C18:1 t11), a hydrogenation intermediate of both C18:2 n-6 and linolenic acid (C18:3 n-3), by the Δ9desaturase in the mammary gland. Increased milk CLA concentrations can
be obtained by inhibiting the rumen biohydrogenation towards increased
CLA c9t11 and C18:1 t11 concentrations. This was previously achieved with
fish oil supplementation (Chilliard et al., 2001). Milk fatty acid composition
can also be optimised by enhancing the concentration of health associated n3 fatty acids, particularly the long chain n-3 fatty acids EPA and DHA which
are present in marine products. Therefore, in vitro and in vivo research with
DHA-enriched micro algae was performed. Firstly, the inhibitory effect of
algae on the in vitro rumen biohydrogenation of C18:2 n-6 was investigated.
Secondly, algae were supplemented to dairy cattle to study their in vivo effect
on rumen and milk fatty acid composition. Additionally, the rumen ciliate
community and the working mechanism of the active algae compound were
investigated to elucidate their effect on the biohydrogenation process and to
further develop strategies for optimising the milk fatty acid composition. The
results of these different studies are synthesised in the present manuscript.
MATERIAL AND METHODS
In vitro incubations
A DHA-enriched algae product was tested in triplicate at four levels (0, 20.8,
41.6, 83.3 mg/incubation) during 24 h in vitro incubations with 25 ml buffered rumen fluid. Sunflower oil (20 mg) and hay (0.4 g) were supplemented
as main source of C18:2 n-6 and as fermentation substrate, respectively.
Total fat content of the different incubations was kept constant by addition
of lard. After incubation, volatile fatty acids, H2 and CH4 were determined
(Fievez et al., 2007). Long chain fatty acids were extracted, methylated and
the amount of fatty acid methyl esters (FAME) was determined by gas chromatography (Vlaeminck et al., 2001).
In a second incubation series, 6 h in vitro incubations with 50 ml buffered
rumen fluid were performed to study the effect of unesterified DHA (21.5
2
mg), either or not supplemented with anti oxidants, on rumen biohydrogenation of C18:2 n-6 from sunflower oil (20 mg). The anti oxidants tested involved the lipid soluble vitamin E (DHA-E; 5 mg DL-all-rac-α-tocopherol) and
the water soluble vitamin C (DHA-C; 5 mg ascorbic acid). After incubation,
FA were extracted, methylated and the amount of fatty acid methyl esters
(FAME) was determined by gas chromatography (Vlaeminck et al., 2001).
In vivo trial
Four multiparous Holstein cows with rumen cannula were used in a 4 x 4
Latin square design during four 3-week periods to evaluate the fatty acid
metabolism in the rumen and milk and the rumen ciliate community. The
current paper is limited to results of two dietary treatments, based on grass
and maize silage, soybean meal, standard dairy concentrate and either or
not supplemented with DHA-enriched algae (Martek DHA-Gold, Schizochytrium sp., 19 % DHA on product basis). Diets were iso-energetic and isonitrogenous. On days 15, 17 and 19 after the start of the algae supplementation (2% of fresh material intake of the previous day), 15 ml of morning and
evening milk were pooled and stored at -20 °C. Likewise, rumen contents
were sampled at various places in the rumen 12 h after morning feeding
(07:00 am) and aliquots of 25 ml were stored in 20 ml of chloroform/methanol (2/1, v/v) at -20 °C. After thawing of milk and rumen content, fatty acids (FA) were extracted, methylated and the amount of fatty acid
methyl esters (FAME) was determined by gas chromatography (Vlaeminck et
al., 2001). Another aliquot of 10 ml rumen content was used for DNA extraction (Griffiths et al., 2000) followed by a nested PCR with general eukaryotic
primers and ciliate specific primers (Boeckaert et al., 2007a). Diversity of the
ciliate community was analysed by Denaturing Gradient Gel Electrophoresis
(DGGE) (7% acrylamide, 35-50% gradient; 38V for 16h at 60°C) and the gels
were processed with BioNumerics software (Boon et al., 2002). The different
ciliates were identified after cloning, sequencing and aligning with the 18S
rRNA gene sequences obtained from the NCBI database.
All statistical analysis were performed using SPPS 12.0.
RESULTS AND DISCUSSION
In vitro effect of algae on rumen biohydrogenation and rumen fermentation
Increasing algae amounts showed only a slight effect on the lipolysis of
C18:2 c9c12 and on the hydrogenation of C18:2 c9t11 (C18:2 c9t11 → C18:1
t11). However, algae DHA provoked a major inhibitory effect on the second
hydrogenation step (C18:1 t11 → C18:0) resulting in the accumulation of
C18:1 t11 (Boeckaert et al., 2007b). Increasing algae concentrations also
resulted in a proportional CH4 inhibition up to 80%, which was associated
with decreased acetate and butyrate and increased propionate proportions
(Fievez et al., 2007).
In vivo effect of algae on the rumen C18 fatty acid composition and rumen
ciliates
Micro algae supplementation to dairy cows provoked significantly higher
concentrations of C18:1 t10 and C18:1 t11 in the rumen (Table 1). This illustrates algae are effective in inhibiting the terminal hydrogenation of C18:1
Comm. Appl. Biol. Sci, Ghent University, 73/1, 2008
3
trans FA to C18:0. Ciliate DGGE profiles showed Isotricha prostoma and
intestinalis and some species of Epidinium caudatum, Eudiplodinium maggii
and Diplodinium dentatum disappeared in the rumen of algae fed cows
(Boeckaert et al., 2007a). Hence, it could be suggested that some ciliates
and/or their associated bacteria play a role in rumen biohydrogenation. This
hypothesis is currently further investigated.
Table 1. In vivo rumen fatty acid composition (g FA/100 g C18 FA) of cows
fed a control versus an algae supplemented diet
CON
ALG
SEM1
D2
D*C3
C18:2 n-6
5.03
7.29
0.48
0.07
0.47
CLA c9t11
0.39
0.16
0.05
0.17
0.21
C18:1 t10
n.d.4
29.1
1.72
**
0.63
C18:1 t11
5.88
28.2
0.82
**
0.09
C18:0
69.0
4.65
0.49
***
0.77
***: P ≤ 0.001; ** : 0.001 < P ≤ 0.01
1SEM: standard error of mean
2D: effect of diet
3D*C: interaction between diet and cow
4n.d.: not detected
Effect of algae on the milk fatty acid composition
The milk of algae supplemented cows showed a significantly lower amount of
saturated FA whereas mono and poly unsaturated FA increased significantly
(Table 2). Moreover, algae supplementation enriched milk fat with the health
associated CLA c9t11 and n-3 FA.
Table 2. Milk fatty acid composition (g FA/100 g FA) of cows fed a control
versus an algae supplemented diet
Control
Algae
SEM1
D2
D*C3
Saturated FA
65.6
53.2
0.864
*
**
Mono unsaturated FA
29.5
36.8
1.031
0.170
**
Poly unsaturated FA
3.16
4.81
0.135
*
*
CLA c9t11
0.48
0.94
0.041
*
*
n-3 FA
0.62
1.62
0.081
*
*
** : 0.001 < P ≤ 0.01; * : 0.01 < P ≤ 0.05
1SEM: standard error of mean
2D: effect of diet
3D*C: interaction between diet and cow
Working mechanism of the algae active compound
Unesterified DHA was found to be the active compound in the micro algae as
illustrated by the significantly higher amounts of hydrogenation intermediates in DHA vs. control incubations (Table 3). Vitamin addition did not prevent DHA to inhibit rumen biohydrogenation as no significant differences
between DHA incubations either with or without vitamin addition could be
observed. Hence, the inhibitory action of free DHA is most probably not provoked by oxidation products of DHA.
4
Table 3. Effect of DHA, either or not supplemented with vit E or vit C, on
the rumen C18 fatty acid composition (g FA/100 g C18 FA)
Control
DHA
DHA-E
DHA-C
SEM
C18:2 n-6
5.33
5.74
6.23
5.50
0.46
CLA c9t11
0.58b
3.02a
3.77a
3.04a
0.25
C18:1 t11
9.82b
17.4a
17.9a
18.3a
0.96
C18:0
61.4a
47.5b
45.0b
46.2b
2.56
a,b Means within a row lacking a common superscript differ significantly
CONCLUSION
The supplementation of DHA-enriched micro algae to dairy cows provokes an
incomplete rumen biohydrogenation resulting in the accumulation of C18:1
trans FA. This is associated with the disappearance of some rumen ciliates
suggesting a possible role of ciliates and/or their associated bacteria in rumen biohydrogenation. Milk fat from algae supplemented cows contains
significantly higher amounts of poly unsaturated fatty acids at the expense
of saturated fatty acids and significantly higher CLA and n-3 FA concentrations could be observed. Anti oxidants could not prevent the inhibitory effect
of unesterified DHA, the active algae compound, on rumen biohydrogenation
which indicates that the latter effect is not due to oxidation of DHA.
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