EFFECTS OF LIPID ENCAPSULATED CONJUGATED
LINOLEIC ACID (CLA) SUPPLEMENTS ON LIPID
METABOLISM IN LACTATING EWES
Eszter GALAMB1, László PÁL1, László WÁGNER1, Ferenc HUSVÉTH1
UČINKI LIPIDNO ENKAPSULIRANIH KONJUGIRANIH
LINOLNO-KISLINSKIH (CLA) DODATKOV NA
PRESNOVO LIPIDOV PRI DOJEČIH OVCAH
ABSTRACT
Several studies have proved that trans-10, cis-12 CLA isomer can induce milk fat depression. In
lactating ruminants the administration of a supplement containing this isomer can be used as a tool
to manipulate milk fat synthesis for reducing negative energy balance in the critical peripartal
period. More studies have been performed with dairy cows, however, only few data are published
with dairy sheep.
The objective of this study was to investigate the effect of a rumen protected form of conjugated
linoleic acid (pCLA) supplement on milk and liver lipid content as well as the fatty acid
composition of milk and liver lipid in lactating sheep.
The animals were divided into two groups. Diet of sheep in the pCLA group was supplemented
with CLA product (equal proportions of cis-9, trans-11 and trans-10, cis-12 isomers) on the other
hand the animals in control group were fed an isocaloric diet supplemented: hydrogenated
triglyceride of palm oil (HTG). In pCLA group we could measure lower total lipid (72.6±5.6 vs.
82.0±6.0) and triglyceride (49.1±5.9 vs. 60.9±5.7) concentrations in the liver samples than in the
HTG group in the peripartal period (d 21 postpartum). Further more we detected higher milk
production (1.89±0.13 vs. 1.73±0.25 kg/d) and milk protein yield (96.2±8.7 vs. 84.9±10.2 g/d)
lower milk fat content (56.8±6.2 vs. 62.7±7.1 g/kg) in the pCLA group. Also the CLA
supplementation influenced significantly the fatty acid profile in milk and liver lipid. The CLA
isomers (cis-9, trans-11 and trans-10, cis-12 C18:2) were increased by CLA supplementation in
the milk lipid. In addition, the pCLA resulted in higher proportions of C18:0, cis-9, trans-11 C18:2
and trans-10, cis-12 C18:2 fatty acids and lower cis-9 C18:1 in the liver lipid comparing to HTG.
We concluded that pCLA supplementation could decrease the risk of lipid accumulation in the
liver of high-lactating dairy ewes in the postpartum period as well as increased the cis-9, trans-11
CLA content of milk lipids; which might have several healthy effects on human consuming sheep
milk or milk products.
KEY WORDS: milk fat depression, conjugated linoleic acid, peripartal period, milk production
1
INTRODUCTION (AND LITERATURE OVERVIEW)
Conjugated linoleic acids (CLA) occur in the milk and meat of ruminant animals and have
numerous favorable physiological effects. It has been demonstrated that cis-9, trans-11
CLA prevents the development of several tumor types induced by specific environmental
1
Department of Animal Science and Animal Production, Georgikon Faculty, University of Pannonia, H8360 Keszthely, Hungary
effects, such as certain skin tumors (Ha et al., 1987; Belury et al. 1996), mammary tumors
(Ip et al., 1991; Thompson, et al., 1997) and gastrointestinal carcinomas (Ha et al., 1990).
Some experimental results prove that CLA isomers have substantial antioxidant effect (Ha
et al., 1990; Ip et al., 1991; Flintoff-Dye and Omaye, 2005). Anti-inflammatory effect of
cis-9, trans-11 CLA was also shown in human cells (Jaudszus et al, 2005). Experiments
were conducted with pigs (Dugan et al., 1997) and mice (Park, et al., 1997) have proved
that supplementation of the feed with CLA markedly reduces the deposition of fat in the
body. The trans-10, cis-12 CLA is associated with diet-induced milk fat depression
(Bauman et al., 2008). In lactating ruminants the administration of a supplement
containing this isomer can be used as a tool to manipulate milk fat synthesis for reducing the
harmful effects of negative energy balance in the critical peripartal period and improving
reproductive performance (Moore et al., 2004; Lock et al., 2006; de Veth et al., 2009).
As CLA isomers are intermediate metabolites of hydrogenation processes of
polyunsaturated fatty acids (PUFA) in the rumen, feeding diets containing ingredients rich
in PUFA, such as oilseeds, vegetable oils (Zhang et al., 2006; Castro et al., 2009) or fresh
grass (Atti et al., 2006) increase CLA content of ruminant products. In the recent years
chemically synthesized and rumen protected CLA supplements are also available which
can be used for the manipulation of milk fat content and fatty acid profile of milk lipids in
lactating ewes or goats (Sinclair et al., 2007; Lock et al., 2008).
The objective of the present study was to study a rumen protected form of CLA
supplement containing approximately equal proportions of cis-9, trans-11 and trans-10,
cis-12 isomers on milk production, milk composition and liver lipid content as well as fatty
acid profile of milk and liver lipids especially C18 fatty acids in lactating Awassi ewes.
2
MATERIAL AND METHODS
2.1
Animals and their management
The experiment was carried out with 50 multiparous lactating Awassi ewes in the farm of
Awassi Co., Bakonszeg, Hungary in compliance with the animal welfare regulations
authorized by the County Veterinary Office (protocol #: DK157/2/2007). The experiment
was conducted according to a complete randomized design from d 2 to 42 postpartum. The
parturition occurred late February 2008. The lambs were weaned immediately after
parturition and the ewes were randomly divided into two groups (25 sheep in both groups)
on the basis of parity, weight and previous lactation performance. Animals were loosehoused in groups on straw bedding in partially covered pens in the same building in
concordant environmental conditions. Experimental diets were formulated according to
the allowances of NRC (2007) and were supplied as total mixed ration (TMR) twice daily
at 7.00 and 19.00 h. Daily amounts of TMR were adjusted to ensure a 10% feed refusal.
The ingredients of TMR is shown in Table 1 (DM: 63.1 %, Crude protein: 14.6 %, NDF:
37.5 %, NE L: 7.1 MJ/kg DM).
Table 1. Ingredient of the experimental ration (TMR) fed of the ewes
Ingredients
Corn silage
Grass hay
Corn grain
Wheat grain
Sunflower meal
Soybean meal
Premix a
Total
a
(g / kg DM )
590
815
465
224
100
246
35
2475
Lactating ewe mineral and vitamine premix, Abomix Ltd., Nyíregyháza Hung; Proct No
Sheep had unlimited access to water and were milked twice a day (6.00 and 18.00 h) by an
automated milking system (De Laval International AB, SE-147 21 Tumba, Sweden).
Before the experimental procedure with the 50 lactating ewes the rumen stability of the
CLA supplement used in the experiment was measured by four adult and non lactating
ewes surgically supplied with rumen cannula (#8C-Rumen; Bar Diamond, Parma, ID,
USA). These animals were kept in free-ranging conditions. However, during the rumen
stability test they were housed in individual cages in a shelter and were fed the same TMR
described earlier. The length of adaptation period to the TMR was 1 week before sampling.
2.2
Experimental design and treatments
The TMR of ewes was supplemented with 25 g/(day x ewe) protected CLA (0.5 MJ NEL).
This product was manufactured by BASF SE (Ludwigshafen, Germany) and marketed as
Lutrell® pure by Arravis Ltd. (Debrecen, Hungary). The supplement was a special
complex of cis-9, trans-11 and trans-10, cis-12 CLA isomers and saturated fat of
vegetable origin and silicic acid. The pCLA contained 788 g lipid and 212 g ash / kg dry
matter. Of the lipid component (as free fatty acid equivalent) 11.8% was the cis-9, trans11 isomer of CLA, 12.1% was the trans-10, cis-12 isomer of CLA, 16.5% was C16:0,
46.0 % was C18:0, 9.8% was C18:1. Supplement provided 2.2 g of both isomers per ewes,
respectively on a daily basis. This amount was very similar to that applied by Lock et al.
(2008) in a study with lactating ewes. The TMR of the ewes in the other group was
supplemented with 21 g of hydrogenated triglyceride of palm oil (HTG; Alifet ® ERBO
Agro AG, 4922 Bützberg, Switzerland) in the same energy content as pCLA. The HTG
contained 951 g fat/kg dry matter (fatty acid composition, g/100g: C12:0 3.2; C16:0 33.7;
C18:0 56.6; C18:1 6.1). At first, both the daily amounts of pCLA and HTG supplements
per group were blended with 2 kg of ground corn grain, respectively and added to the
TMR of ewes in two equal proportions before feeding. Experimental diets were fed from d
2 to 42 postpartum.
Collection of samples
Rumen stability of pCLA was measured by the in situ method of Ørskov and McDonald
(1979), originally developed for determining protein degradation in the rumen and
modified in our laboratory to measure the rumen degradability of feed supplements
stabilized by saturated vegetable fat encapsulation (Elek and Husveth, 2007).
In the experiment with lactating ewes samples of TMR were collected weekly for analysis.
The amount of feed refused (orts) was recorded daily per group and collected weekly, and
sampled for analysis. The dry matter intake (DMI) of the groups was calculated daily by
the difference between feed intake offered and feed DM refused. Data were grouped
weekly and these weekly subsets were evaluated statistically.
The individual milk production was recorded daily and milk samples were taken at both
the morning and afternoon milking by Tru Test (Auckland, New Zealand) milk meter on d
7, d 14, d 21, d 28, d 35 and d 42. Morning and evening milks were mixed in
representative proportions and were homogenized by vortexing them for 30 sec. The
homogenized samples were frozen to - 20 ºC and stored for fat and protein analyses and
fatty acid analysis.
At 2 d postpartum (i.e. at the beginning of the experiment) 10 animals were selected
randomly from both pCLA and HTG groups, respectively. At d 2, 21 and 42 postpartum
liver tissue samples (100-150 mg) were collected by percutaneous biopsy from the 11th or
10th intercostal space using an aspiration needle of 3-mm diameter (Vekas Ltd., Debrecen,
Hungary) under local anesthesia with 3-5 ml of 2 % lidocaine. The animals selected before
and during the experiment were always the same. Immediately after collection liver
samples were weighed by an analytical scale and then stored in a well-sealed plastic tube
on ice until the analysis.
Laboratory procedures
Feed and orts samples were analyzed for dry matter content (DM), crude protein (CP),
ether extract and ash by the methods 934.01, 976.05, 920.39, and 942.05 of the AOAC
(2000), respectively. Neutral detergent fibre (NDF) was determined according to van
Soest et al. (1991) using hot ethanol, 8 M urea, and heat stable α-amylase (Sigma A3306;
Sigma Chemical Co., St. Louis, MO, USA). Ash content was excluded from the NDF.
Following melting and homogenization (vortexing for 30 sec) of milk samples, total fat
content was determined by the method of Folch et al. (1957). The lipid phase of samples
was extracted by a 2:1 mixture of chloroform-methanol. In the second step, the filtrate was
separated from non-lipid structure by washing it with saline water. For the milk protein
analysis the nitrogen content was measured by the Kjeldahl method (991.22 AOAC, 2000).
Milk crude protein was estimated as N (%) x 6.38.
Liver samples were also extracted by the method of Folch et al. (1957). Liver biopsy
samples was homogenized with 10 ml of a 2:1 (vol/vol) mixture of chloroform-methanol
after which 2.0 ml 0.9 % NaCl was added, mixed and let to stand for 2 h to allow phase
separation. The chloroform-methanol extract was evaporated to dryness in a water bath at
50 °C under N2 flow then the residue was weighed in an analytical scale for TL content of
the sample and expressed as g/kg of wet liver tissue. Based on the lipid weight, the dry
extracts were dissolved in different volumes of isopropanol (range 2 to 8 ml; Piepenbrink
and Overton, 2003) then they were analysed for triglyceride content (TGL) using a
commercial test kit (Reanal Fine Chemicals Co., Budapest, Hungary; intra- and interassay
CV %: 2.1/6.4) with isopropanol as a blank.
Fatty acid compositions were determined of extracted milk and liver lipid samples. After
the extractions of lipids from milk or liver tissue described above, lipid extracts were
carried in a screw-cap test tube for the hydrolysis of fats, and 1 mg of nonadecanoic acid
(C19:0) as an internal standard was added to the samples. Methylation of the samples was
prepared using tetramethylguanidine (TMG) and methanol, as described by Phillip et al.
(2007). Fatty acid methyl esters were analyzed using a TRACE type gas chromatograph
(Model 2000; Thermo Finnigan Italia S.P.A., 2009 Rodano, Milan, Italy) equipped with
SSL injector and flame ionization detector. For the separation of fatty acid methyl esters a
capillary column (100 m × 0.25 ID) coated with AP 2560 (Supelco Inc., Bellefonte, PA,
USA) was used. During chromatography, helium was used as carrier gas at a constant
pressure of 200 kPa. The automatically regulated temperature of the capillary tube varied
between 150 and 215 °C during the measurement. The total time required for the
chromatography of one sample was 75 minutes. Identification of the peaks obtained was based
on retention times determined using a standard mixture of fatty acids with known composition
(Matreya, Inc. Pine Hall Drive, State College, PA, USA; Catalogue No. 1254-7).
For fatty acid analysis of dietary components, total lipid was obtained by boiling the
samples in 4 M HCl for 1 h before extracting the lipids with diethyl ether (method 920.39
AOAC, 2000). Further steps of the analysis were similar to those applied for milk and
liver samples with the exception that methyl esters of fatty acids were separated with an
Omegawax 320 capillary column (30 m length; Supelco Inc., Bellefonte, PA, USA).
Statistical analysis
The data were analyzed by the GLM procedure of SPSS 9.0 (SPSS software for Windows,
release 9.0, SPSS Inc., Chicago, IL, USA). First, data were tested by KolmogorovSmirnov test for normal distribution. ANOVA were performed on normally distributed
data using dietary as a fixed main effect and sampling time as repeated measures.
Statistical significant differences were determined at P<0.05; however, 0.05<P<0.10
indicated a trend for differences between treatments.
3
RESULTS AND DISCUSSION
Rumen stability of the CLA isomers in the pCLA supplement used in the experiment and
tested by rumen fistulated ewes are demonstrated. After 8 h of incubation in the rumen
undegradable proportions of c9, t11 CLA and t10, c12 CLA were 72.9 % and 76.7 %,
respectively, and showed no values lower than 50 % for either isomer, even at 24 h
incubation period.
Data of DMI and milk production are presented in Table 2. Dietary treatments had no
considerable effects on the DMI of the ewes. However, sheep fed diet supplemented with
pCLA showed higher daily milk production (P < 0.05) and milk protein yield (g/day; P <
0.01) but lower milk fat content (g/kg milk; P < 0.01) than those fed the HTG diet. No
significant differences were found in daily milk fat yield or milk protein content between
the treatments.
In several studies with lactating cows (Perfield et al. 2002; Piperova et al, 2004) or with
lactating ewes (Sinclair et al., 2007) no effects of CLA supplementation were observed on
milk yield, milk protein concentration and yield or lactose content. However, BernalSantos et al. (2003) found that cows fed CLA tended to produce approximately 3 kg/d
more milk during the first 20 wk of lactation than control animals without CLA
supplementation. In the study by Lock et al. (2006) 2.4 g/(day x ewe) lipid-encapsulated
trans-10, cis-12 CLA addition to the diet of lactating ewes resulted in an about 10 %
increase in milk yield and 7 % increase in milk protein yield. These results are quite similar
to those found in our study, when 2.2 g was applied from this CLA isomer in the diet.
Table 2. Effect of protected CLA (pCLA) supplementation on dry matter intake (DMI),
milk production and milk composition of lactating ewes (means ± SD, n=25)
Diet
a
g
DMI (kg/d)
Milk yieldh (kg/d)
Milk fat
g/kg
g/day
Milk protein
g/kg
g/d
b
c
Significancef
DIMd Trt x DIMe
ns
ns
***
ns
pCLA
2.45±0.35
1.89±0.13
HTG
2.38±0.28
1.73±0.25
Trt
ns
*
56.8±6.2
107.3±10.1
62.7±7.1
108.5±16.2
**
ns
**
ns
ns
ns
50.9±5.8
96.2±8.7
49.1±3.7
84.9±10.2
ns
**
ns
ns
ns
ns
a
25g (0.5 MJ) pCLA/kg of diet DM
21g (0.5 MJ) HTG/kg diet DM
c
Trt: treatment effect; dDIM: days in milk; eTrt x DIM: treatment effect and days in milk interaction
f
Significance between data within the same know; ns: nonsignificant; *: P<0.05; **: P<0.01; ***: P<0.001
g
DMI: dry matter intake
h
Data of milk production and composition are the average of the 5 week experimental period
b
The studies above proved that CLA supplements containing trans-10, cis-12 isomer
significantly decrease milk fat content. In our study milk yield of ewes fed a diet
supplemented with pCLA increased almost 10 % on an average. At the same time CLA
decreased milk fat content by 9.4 %. As a result of the reverse effects of CLA
supplementation on milk yield and milk fat content. These data are consistent with the
results of Bernal-Santos et al. (2003) found in dairy cows. In their study, despite the fact
that the milk fat percentage was decreased by the CLA supplementation, yield of 3.5 % fat
corrected milk did not show significant differences between cows fed diets supplemented
by CLA or Ca salts of palm fatty acid distillate. They reported that dairy cows with CLA
treatments responded by partitioning more nutrients toward milk synthesis rather so
improving energy balance. We think that in our experiment similar process was occurred.
Probably the energy precursors taken up by the mammary gland for fat synthesis did not
change. However more nutrients were utilized for the synthesis of milk components other
than milk fat including protein.
Fatty acid content of milk lipids are summarized in Table 3. As significant differences
were noticed only in some C18 fatty acids, this table shows only the results concerning
these fatty acids. Rumen protected CLA supplementation caused about two-fold increase
in cis-9, trans-11 CLA and five-fold increase in trans-10, cis-12 CLA isomer contents in
the milk compared to the milk of HTG ewes. The rate of increase was quite similar to
those observed in the studies with lactating ewes fed on diets supplemented with rumen
protected supplement rich in trans-10, cis-12 CLA (Lock et al., 2006; Sinclair et al., 2007).
The proportion of cis-9, trans-11 CLA in the milk and the rate of increase in this fatty acid,
relative to the HTG treatment, was somewhat higher than those reported by the other
researchers indicated above. This result shows that cis-9, trans-11 CLA in the pCLA
applied in our experiment can be used to increase the proportion of this CLA isomer in the
milk. This can be one of the tools to produce milk with healthier fatty acid composition in
dairy sheep, too.
Table 3. Effect of supplementation with protected CLA (pCLA) on C18 fatty acid
composition (g/100 g FA) in milk lipids (means ± SD, n=25)
Fatty acids
c9 C18:1
c9, t11 C18:2
t10, c12 C18:2
pCLAa
23.08±3.00
1.62±0.34
0.71±0.11
HTGb
24.07±2.92
0.82±0.12
0.14±0.09
Trtc
ns
***
***
Significancef
DIMd Trt x DIMe
*
ns
ns
ns
ns
ns
a
25g (0.5 MJ) pCLA/kg of diet DM; b21g (0.5 MJ) HTG/kg diet DM; cTrt: treatment effect; dDIM: days in
milk; eTrt x DIM: treatment effect and days in milk interaction; fSignificance between data within the same
know; ns: nonsignificant; *: P<0.05; **: P<0.01; ***: P<0.001
The relatively high cis-9, trans-11 CLA content in the milk lipids of the HTG ewes in this
experiment shows, however, that either microbial synthesis in the rumen or the production
by 9-desaturase from trans-11 C18:1 in the mammary gland are also important sources
for milk cis-9, trans-11 CLA. Interestingly, the cis-9 C18:1 contents of the milk samples
collected at 35 DIM was lower (P < 0.05) than those in the samples collected at the earlier
sampling times (7, 14 and 21 DIM). In the earlier period of lactation lipid mobilization
due to negative energy balance might have a significant effect on the fatty acid
composition of milk lipids. In this period cis-9 C:18:1 fatty acid proportion was proved
higher in both plasma or other tissues according to the higher mobilization rate of this
fatty acid from the adipose tissue (Smith and Walsh, 1975; Karcagi et al., 2008).
In our experiment the observation of Lock et al. (2006) that fatty acid profile in the milk
of lactating ewes fed on a diet supplemented with CLA shifts toward an increased
proportion of long-chain fatty acids, was not found. This difference can be perhaps
explained by the difference in the stage of lactation at which the treatment was applied.
The experimental period in our research started immediately after lambing, however, that
of the authors mentioned before started at approximately 5 wk postpartum. This
hypothesis is supported by the findings of Bernal-Santos et al. (2003). In their study trans10, cis-12 CLA supplementation did not affect milk fat content until several weeks after
the initiation of lactation and there was no shift in milk fatty acid composition until several
weeks postpartum.
Total lipid and triglyceride content of the liver showed differences between dietary
treatments and sampling times in our experiment (Tab. 4). Relatively high initial (at d 2
postpartum) TL and TG contents were measured in the liver of the ewes which further
increased by the d 21 postpartum, then decreased to low concentrations by d 42
postpartum. These findings are similar to those observed by Smith and Walsh (1975) in
peripartal Masham ewes in the first pregnancy or lactation. These researchers showed the
highest TG values at d 14 postpartum which proved significantly higher than at d 140 of
pregnancy. In the present study dietary treatments resulted in differences of liver TL or
TG contents only at d 21 postpartum when the highest liver lipid content was measured.
At this sampling time CLA supplementation decreased both TL and TG concentrations in
the liver.
Table 4. Effect of supplementation with protected CLA (pCLA) on total lipid (TL) and
triglyceride (TG) content (g/kg) in the liver of ewes
Trtc
Significancef
Dppd
Trt x Dppe
66.3±10.4
72.6±5.6
82.0±6.0
51.1±8.3
48.8±7.3
*
ns
***
***
ns
ns
37.9±8.7
49.1±5.9
60.9±5.7
24.8±3.0
24.6±5.4
**
ns
***
***
ns
ns
Diet
pCLAa
TL (dpp)g
2h
21
42
TG (dpp)g
2h
21
42
HTGb
a
25g (0.5 MJ) pCLA/kg of diet DM; b21g (0.5 MJ) HTG/kg diet DM; cTrt: treatment effect; dDpp: effect of
days postpartum; eTrt x Dpp: treatment effect and days postpartum interaction; fSignificance between data
within the same know; ns: nonsignificant; *: P<0.05; **: P<0.01; ***: P<0.001; gDpp: days postpartum;
h
at the beginning of the experiment
More observations prove that liver lipid content increase in high yielding dairy ruminants
in the peripartal (transition) period is partially due to greater fatty acid uptake of the liver
(Emery et al, 1992). Fatty liver occurs when the rate of hepatic triglyceride (TG) synthesis
exceeds the rate of TG disappearance through either hydrolysis or secretion via very lowdensity lipoproteins (VLDL). The rates of hepatic TG synthesis are similar between
ruminants and nonruminants, but for unknown reasons, ruminants have a very slow rate of
hepatic VLDL secretion relative to most species (Pullen et al., 1990). Serum concentration
of apolipoprotein B100, the major apolipoprotein of VLDL, was found to be lower in
cows experiencing metabolic disorders such as ketosis or fatty liver (Itoh et al., 1997). As
VLDL-lipoprotein synthesis is limited (Kleppe et al., 1988; Emmison et al., 1991) it is
resulting in a lower TG export from the liver.
The lower TL and TG accumulation in the liver of the ewes fed the diet supplemented
with pCLA in our experiment can be perhaps explained by the higher excretion of
lipoproteins from the liver which is low in ruminants. The effect of pCLA on ruminant
lipoprotein metabolism has not been studied so far, but this hypothesis may be supported
by the study of Liu et al. (2007) who showed higher production rate of VLDL-TG in the
liver of mice treated with CLA.
Fatty acid composition of liver lipids is presented in Table 5. Both at d 21 or d 42
postpartum the proportions of two CLA isomers were higher (P < 0.001) in the liver lipids
of CLA-fed ewes than those of HTG group, respectively. Lower cis-9, trans-11 CLA
contents were detected in the liver of ewes than those in the milk lipids fed the diet
supplemented with either pCLA or HTG. These results also show that the mammary
synthesis of this isomer is a major source in the lactating ewes. Contrary to cis-9, trans-11
CLA changes higher proportions of trans-10, cis-12 CLA were detected in the liver lipids
than in milk. This finding show that the latter fatty acid incorporates into the milk lipids in
a lower rate, however, it accumulates well into the liver lipids.
Table 5. Effect of supplementation with protected CLA (pCLA) on C18 fatty acid
composition (g/100 g FA) of liver lipids in lactating ewes (means ± SD, n=10)
2c
Fatty acids
C18:0
c9 C18:1
c9, t11 CLA
t10, c12 CLA
19.50±2.41
23.69±1.52
0.29±0.08
0.40±0.16
Days postpartum (dpp)
21
42
a
b
a
pCLA
HTG
pCLA
HTGb
d
e
15.77±2.25 13.94±1.21
22.6±1.52
23.18±1.89
d
e
23.34±2.10 26.52±2.18
14.46±2.67 16.35±2.01
0.58±0.11d
0.22±0.16e
0.64±0.08d
0.31±0.11e
0.77±0.27d
0.47±0.14e
0.86±0.18d
0.53±0.13e
a
25g (0.5 MJ) pCLA/kg of diet DM; b21g (0.5 MJ) HTG/kg diet DM; cat the beginning of the experiment
Pairwise comparison: means in the same row and the same sampling time marked significant differences
P<0.001
d,e
Significant differences were also observed in the C18:0 and c9 C18:1 content of the liver
lipids. At d 21 postpartum, when liver TL or TG concentrations were higher than those
observed at d 2 or 42 postpartum C18:0 showed higher (P < 0.05), however cis-9 C18:1
showed lower (P < 0.01) values in the pCLA group than in the HTG. Stearic (C18:0) and
oleic (C18:1) acid changes in the liver lipids reflect well the TL and TG changes published
in lactating ruminants in the peripartal period (Smith and Walsh, 1975; Husveth et al.,
1982; Rukkwamsuk et al., 1999). At d 21 postpartum, when the liver TL and TG
concentrations were higher than d 2 or d 42 postpartum, proportion of C18:0 was lower
and that of C18:1 was higher than at the other two sampling times. Decreasing liver lipid
concentration pCLA resulted in higher C18:0 and lower C18:1 proportions relative to
HTG at d 21 postpartum, which are shown typical in the liver of cows with lower fatty
infiltration in the peripartal period.
4
CONCLUSION
Dietary application of rumen protected CLA supplement containing approximately equal
quantities of cis-9, trans-11 CLA and trans-10, cis-12 CLA isomers increases milk
production but decreases milk fat content in the early lactation period in ewes. The lower
milk fat content associates with lower liver TL and TG concentration in the period when
the energy balance generally negative induced by the high energy requirement of milk
production. From this reason dietary CLA supplementation containing trans-10, cis-12
isomer can decrease the risk of lipid accumulation in the liver of high lactating dairy ewes
in the postpartum period. The other isomer of the supplement efficiently increases the cis9, trans-11 CLA content of milk lipids which may have favorable health effects for
humans consuming sheep milk or milk products.
5
SUMMARY
The effect of a rumen protected form of a conjugated linoleic acid (pCLA) supplement
containing approximately equal proportions of cis-9, trans-11 and trans-10, cis-12 isomers
(11.8 and 12.1 % wt/wt as free fatty acid equivalents) on milk production, milk
composition and liver lipid content; as well as the fatty acid profile of milk and liver in
lactating sheep was studied. Fifty multiparous Awassi ewes were randomly divided into
two equal groups after parturition and were fed a total mixed diet of corn silage, grass hay
and concentrate from d 2 to 42 postpartum. Diet of ewes in the pCLA group was
supplemented with 25 g/(day x ewe) of pCLA providing 2.2 g/(day x ewe) of both CLA
isomers while sheep in the control group were fed an isocaloric diet supplemented with 21
g of a hydrogenated triglyceride of palm oil (HTG group). Milk yield was measured daily
and milk composition was analysed on a weekly basis. Liver total lipid, triglyceride and
fatty acid composition was determined from liver samples collected by percutaneous
needle biopsy d 2, d 21 and d 42 postpartum. Higher milk production (1.89±0.13 vs.
1.73±0.25 kg/d) and milk protein yield (96.2±8.7 vs. 84.9±10.2 g/d) and lower milk fat
content (56.8±6.2 vs. 62.7±7.1 g/kg milk) were detected in the pCLA sheep than in the
HTG group. No differences were observed in the dry matter intake, milk fat yield or milk
protein concentration between the two groups. Supplementation with pCLA resulted in
lower total lipid (72.6±5.6 vs. 82.0±6.0) and triglyceride (49.1±5.9 vs.60.9±5.7)
concentrations in the liver samples collected d 21 postpartum than in the HTG group. The
pCLA supplementation increased cis-9, trans-11 C18:2 and trans-10, cis-12 C18:2, and
decreased cis-9 C18:1 fatty acid contents in the milk lipids. In the liver lipids resulted
pCLA supplementation in higher proportions of C18:0, cis-9, trans-11 C18:2 and trans-10,
cis-12 C18:2 fatty acids and lower cis-9 C18:1 comparing to HTG. We conclude that
containing trans-10, cis-12 isomer of pCLA supplementation could decrease the risk of
lipid accumulation in the liver of high-lactating dairy ewes in the postpartum period. The
other isomer of pCLA increased the cis-9, trans-11 CLA content of milk lipids, which
may have favorable health effects for humans consuming sheep milk or milk products.
ACKNOWLEDGMENT
This study was financially supported by the National Scientific Research Fund of Hungary
(OTKA) project # K 68779.
6
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