Transcript Lipid Metabolism In Ruminants

Lipid Metabolism In
Ruminants
G. R. Ghorbani
Overview
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Herbivores diets are normally quiet low in lipid
because of the small quantity (2-5%) contained in
most plant food sources.
These dietary characteristics have required both
metabolic adaptations and methods for conserving
essential fatty acids (EFA).
Plant lipids are altered extensively by the rumen
fermentation, and the lipid actually received and
absorbed by the animal differs from that ingested.
Overview
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The rumen is intolerant to high levels of fat,
which may upset the fermentation.
This situation in functioning ruminant contrast
with that in the newborn ruminant, which
ingests milk at about 30% or more fat in the
DM, representing 50% or more of its caloric
intake.
Overview
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In most metabolic systems FA’s are derived
from glucose.
Dietarily derived glucose is scarce in
ruminant metabolism, however and ruminants
have evolved mechanisms for its
conservation, the most important of which is
the lack of pathways for converting glucose
into FA’s.
Overview
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About 90% of fat synthesis in ruminants
occurs in the adipose tissue.
The liver, which is the major lipogenesis site
in many non-ruminants species, accounts for
only 5% in ruminants.
Plant Lipids
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Lipids can be grouped into storage compounds
in seeds (TG), leaf lipids (galactolipids and
phospholipids), and a miscellaneous assortment
of waxes, carotenoids, chlorophyll, essential oils
and other ether-soluble substances.
TG are negligible in forages.
Leaf lipids are mainly galactolipids involving
glycerol, galactose, and unsaturated FA’s.
Plant Lipids
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The leaf lipids are generally more polar than
TG’s and have a lower energy value than
would be estimated by the 2.25 factor used to
calculate TDN.
The FA’s associated with GL and many of the
TG’s of the seed organs are relatively
unsaturated and contain high amounts of
linoleic and linolenic acids (Table 1).
Table-1. content and composition of EE from forage
leaves
% Of DM
% of EE
Ether extract
5.3
100
Fatty acids
2.3
43
Wax
0.9
17
Chlorophyll
0.23
4
Galactose
0.41
8
1.0
19
Non fatty acid
unsaponifable fat
Triacylglycerol
Triglycerides
R-COO-CH2
R-COO-CH
R-COO-CH2
• Triglycerides found in seeds and
animal adipose.
• Diglycerides found in plant leaves,
one fatty acid is replaced by a sugar
(galactose).
Triglyceride Containing Linoleic Acid
Omega-6
Linolenic Acid
Omega-3
Fatty Acid Isomers
Lipolysis
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Shortly after esterified plant lipids are
consumed, they are hydrolyzed extensively by
microbial lipases, causing the release of
constituent FA’s.
Anaerovibrio lipolytica , which is best known for
its lipase activity, produces a cell bound
esterase and a lipase.
The lipase is an extracellular enzyme packaged
in membranous particles composed of protein,
lipid, and nucleic acid.
Lipolysis
+ 3H20
+
Lipases
Esterified Plant
Lipid
Free Fatty Acids
Lipid Digestion
Rumen
-galactosidase
DigalDigly
MonogalDigly
-galactosidase
Galactose
Propionate
Diglyceride
Lipase Anaerovibrio
lipolytica
Glycerol
Triglyeride
CaFA
Lipase
Fatty acids
Saturated FA
Ca++
H+
Reductases
Feed particles
Lipolysis
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The lipase hydrolyzes acylglycerols completely
to FFA, glycerol and galactose with little
accumulation of mono or diglycerides.
Glycerol and galactose are fermented rapidly,
yielding propionic and butyrate acid as a major
end product.
Despite its high lipase activity, the general
esterase activity in A.lipolytica is lower than in
many non lipolytic bacteria.
Hydrolysis of lipids in the rumen
(Bath & Hill 1969)
% of Total Lipid
Total Lipid
%
TG
DG
MG
PL
FFA
Diet*
6.3
72.4
13.7
1.7
1.2
11
Rumen digesta, 0 h
5.1
.1
.0
.1
15.2
85
Rumen digesta, 1 h
6.2
30.4
1.7
.0
7.1
61
Rumen digesta, 5 h
6.4
11.1
.0
.0
12.4
76
*Diet consisted of 1 kg chopped hay + 50 g of palm oil
Lipolysis
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Fay et al identified 74 strains of ruminal
bacteria that were capable of hydrolyzing the
ester bond.
Known lipolytic strains, including A lipolytic,
and Butyrivibrio fibrosolvens, had low
hydrolysis in that assay.
Also, bacteria with general esterase activity are
not necessarily capable of hydrolyzing lipid
esters.
Hespell and O’Bryan-shah found a wide variety
of ruminal bacteria with esterase activity,
including 30 strains of B. fibrosolvens, but only
a few bacteria could hydrolyze long chain fatty
acids (LCFA)
Lipolysis
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The extent of hydrolysis is very high for most
unprotected lipids: 85-95%.
This % is higher for diets rich in fats than for
conventional diets, in which most lipids are in
cellular structures.
Hydrolysis seems to be highest for diets rich
in protein.
Lipolysis
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Gerson et al. have shown that lipase was more
active with diets rich in fiber than for diets rich
in starch, but that a short-term supply of starch
in a fiber diet could increase lipolysis.
This suggests either that the rate of lipolysis
could depend on the microbial ecosystem, or
that variations of ruminal pH control lipase
activity.
Lipolysis
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Protozoa are not involved to any great extent
in hydrolysis, except for that of phospholipids.
Salivary lipase present in ruminants has a
very low activity, whereas in monogastric
animals it plays a more important role
Biohydrogenation
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Unsaturated FFA have relatively short half
lives in ruminal contents because they are
rapidly hydrogenated by microbes to more
saturated end products.
The initial step in biohydrogenation (BH) is an
isomerization reaction that converts the cis12 double bond in unsaturated FA’s to a
trans-11 isomer.
Biohydrogenation
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Reduction of double bonds
Result: fatty acids that are more saturated
with hydrogen
Unsaturated
Saturated
Linolenic Acid
Omega-3
Hydrogenation of Fatty Acids
in the Rumen
Polyunsaturated fatty acids (all cis)
Isomerase (from bacteria)
Needs free carboxyl group
and diene double bond
Shift of one double bond (cis & trans)
Hydrogenation
Hydrases (from bacteria,
Hydrogenated fatty acid
(stearic and palmitate)
mostly cellulolytic)
Hydrogenation of Fatty Acids
in the Rumen
All unsaturated fatty acids can be hydrogenated
Monounsaturated less than polyunsaturated
65 to 96% hydrogenation
Numerous isomers are produced
Biohydrogenation is greater when high forage
diets fed
Linoleic acid depresses hydrogenation of FA
Biohydrogenation
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The isomerase is not functional unless the FA
has a free carboxyl group, and in the case of
PUFA;s such as C18:2, a cis-9, cis-12 diene
double bond configuration is present.
The requirement of a free carboxyl group
establishes lipolysis as a prerequisite for
biohydrogenation .
Biohydrogenation
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Once the trans-11 bond is formed by action of
the isomerase, then hydrogenation of the cis-9
bond in C18:2 occurs by a microbial reductase.
The extent to which trans-11 C18:! Is
hydrogenated to C18:0 depends on conditions in
the rumen.
For example, complete hydrogenation to stearic
acid is promoted by the presence of cell-free
ruminal fluid and feed particles, but it is inhibited
irreversible by large amounts of linoleic acid.
Biohydrogenation
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Linolenic acid is often completely
hydrogenated in stearic acid.
The hydrogenation of linleic acid is not
complete..
It provides stearic acid and different
monounsaturated isomers, of which transvaccenic acid is characteristic of ruminal
metabolism
Biohydrogenation
t11 18:1
con. 18:2
18:2
18:0
18:2 converted
(%)
100
80
60
40
20
0
0
1
2
3
Time (h)
(adapted from Harfoot et al., 1973)
4
5
6
Conjugated Linoleic Acid - Rumen
Most Common Pathway (High Roughage)
Linoleic acid (cis-9, cis-12-18:2)
Cis-9, trans-12 isomerase
Butyrivibrio fibrosolvens
Conjugated linoleic acid (CLA, cis-9, trans-1118:2)
Vaccenic acid (Trans-11-18:1)
Stearic acid (18:0)
At low rumen pH, trans-10, cis-12 isomer of CLA
is produced.
CLA absorbed from the intestines available
for incorporation into tissue tryglycerides.
Reactions from linoleic acid to vaccinic acid
occur at a faster rate than from vaccinic acid
to stearic acid.
Therefore, vaccinic acid accumulates in the
rumen and passes into intestines where it
is absorbed.
Quantities of vaccinic acid leaving the rumen
several fold greater than CLA.
Conversion of Vaccinic Acid to CLA
In mammary gland and adipose
Trans-11-18:1
CLA, cis-9, trans-11 18:2
Stearoyl CoA Desaturase
‘9-desaturase’
This reaction probably major source of CLA in
milk and tissues from ruminants.
Also transforms
Palmitic
Stearic
Palmitoleic
Oleic
CLA Isomers - Rumen (High Concentrate)
Low Rumen pH
Linoleic acid (cis-9, cis-12-18:2)
Cis-9, trans-10 isomerase
CLA Isomer (trans-10, Cis-12-18:2)
This isomer is inhibitory to milk
fat synthesis.
Trans-10-18:1
Effect of CLA isomers on milk fat
%
Milk Fat, percentage
Infusion
3.5
3
c/t 10,12 CLA
c/t 9,11 CLA
Control
2.5
2
1.5
-2
-1
1
2
3
4
5
6
7
8
Day
Baumgard et al. (2000)
Potential Value of CLA in
Foods of Ruminant Origin
Anticarcinogenic effects in lab
animals given chemicals to cause cancer
Reduce atherosclerosis
Direct evidence with rabbits
Indirect evidence with humans
Reduce fat accumulation in the body
Laboratory animals and pigs
Evidence not conclusive with humans
Effect of dietary supplementation of
CLA or SAFF on lipid concentration
(%) in various tissues
Muscle
Diaphragm
Leg
Adipose
Liver
CON
CLA
SAFF
SEM
26.9
12.8
31.5
13.9
25.0
14.2
1.88
1.18
b
71.7
13.6a
c
58.7
a
82.6
3.00
11.1b
13.5a
0.77
CLA Content of Foods
Food
CLA isomers
mg/g fat
Beef
Pork
Chicken
Milk
Colby cheese
Corn oil
4.3
0.6
0.9
5.5
6.1
0.2
cis 9, trans 11
%
85
82
84
92
92
39
Partially Hydrogenated Vegetable Oil (PHVO)
Microbial Fatty Acid Synthesis
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Total lipid content of bacterial dry mass in the
rumen ranges from 10 to 15%.
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Bacterial lipids originate from exogenous
sources (uptake of dietary LCFA) and
endogenous (de novo synthesis) sources; the
contribution of each source depends on lipid
content of the diet and bacterial species.
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Increasing lipid concentration in the diet
enhance exogenous uptake by some microbes.
Microbial Fatty Acid Synthesis
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FA’s synthesized de novo consist mainly of
C18:0 and C16:0 in an approximate ratio of 2:1
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Significant amounts of radioactivity from [14C]
acetate or [14C] glucose are incorporated into
microbial lipid as straight chain, even-numbered
carbon fatty acids.
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Propionate or valerate substitute for acetate
yields straight chain, odd numbered carbon
LCFA in ruminal microbes.
Microbial Fatty Acid Synthesis
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Branched chain FA’s (Iso) can be accounted for
by utilization of isobutyrate, isovalerate, and 2methylbutyrate as primers.
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Monounsaturated FA’s that constitute 15 to 20%
of bacterial FA’s are synthesized by the
anaerobic pathway.
Microbial Fatty Acid Synthesis
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Polyunsaturated FA’s are not commonly synthesized
by bacteria.
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Therefore, PUFA’s reported to exist in ruminal
microbes are likely the result of exogenous uptake
of preformed FA’s.
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Odd numbered FA can be obtained by reducing the
chain length through alpha-oxidation or from
propionyl-CoA.
Fatty Acid Composition (% by weight) of
Lipids of Mixed Rumen Bacteria
% Composition
Fatty Acid
11:0
12:0
12:0br
13:0
13:0br
14:0
14:0br
15:0
15:0br
16:0
Cattle
Sheep
0.1
0.4
0.7
0.3
0.7
2.3
2.4
4.4
10.1
35.2
n.d.
4.6
n.d.
n.d.
n.d.
3.7
n.d.
n.d.
n.d.
25.4
Fatty Acid Composition (% by weight) of
Lipids of Mixed Rumen Bacteria
% Composition of
Cattle
Sheep
1.0
-1.8
1.7
32.0
n.d
3.9
3.5
n.d.
tr
n.d
n.d.
20.8
n.d
19.7
5.6
tr
20.2
Fatty Acid
16:0br
16:1
17:0
17:0br
18:0
18:0br
18:1
18:2
18:3
20:0
--
Lipid Balance Across The Rumen
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Fatty acids loss from ruminal content was
negligible in many studies that examined
LCFA absorption across the ruminal
epithelium or their catabolism to VFA or CO2.
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85 to 96% of radioactive linoleic acid added
to the rumen of sheep was recovered from
ruminal contents after 48 h.
Lipid Balance Across The Rumen
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Likewise, radioactivity was minimal in blood
plasma of sheep given a ruminal dose of labeled
unsaturated LCFA.
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Degradation of LCFA to CO2 and VFA was less
than 1% when acids were incubated with ruminal
microbes in vitro or in vivo, demonstrating that
LCFA have little energy-sparing effect on growth
of ruminal microbes.
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Protozoa, especially holotriches, ingest LCFA
mainly for direct incorporation into cellular
lipids, but few LCFA are catabolized.
Possible Routes of FA Loss From
Ruminal Fluid
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Goosen incubated [14C]oleic acid with ruminal epithelium
and reported 31.5% uptake by the tissue and 8.2%
transport.
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Plamitate was metabolized readily to KB’s by ruminal
epithelium and to C15 acids by  oxidation and then to
C13 and C11 acids by ß oxidation.
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Palmitate oxidation, and its conversion to KB’s also
occurred in epithelial cells isolated from the rumen of
sheep.
Possible Routes of FA Loss From
Ruminal Fluid
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Wu reported greater than 90% disappearance
from the rumen of FA’s shorter than C14.
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Table 1 summarized data from 15 published
studies that examined lipid flow to the small
intestine of cattle or sheep. Of the 47 animal
groups, 15 had net loss of lipid from the mouth
to duodenum. Lipid loss across the rumen was
more common for diets with added fat (11 out of
15) than for control diets (4 out of 15).
Possible Routes of FA Loss From
Ruminal Fluid
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Regression of dietary lipid flow against lipid
intake (Figure 3) gave a slope of .92,
indicating loss of dietary lipid in the rumen
equal to 8 g/100 g of lipid intake.