Published December 3, 2014
Effect of linseed addition on the expression of some lipid metabolism
genes in the adipose tissue of young Italian Simmental and Holstein bulls1
M. Corazzin,* S. Bovolenta,*2 E. Saccà,* G. Bianchi,† and E. Piasentier*
*Department of Agricultural and Environmental Science, University of Udine, 33100 Udine, Italy; and †ERSA, Agency for
Rural Development, Autonomous Region of Friuli Venezia Giulia, 33100 Udine, Italy
were similar (P ≥ 0.23) between bull breeds, s.c. fat
from Holstein bulls had greater (P < 0.05) proportions of
tridecylic (C13:0), myristoleic (C14:1) and palmitoleic
(C16:1n-9cis) acids and a lower (P < 0.05) proportion of
margaric (C17:0) acid than s.c. fat from Simmental bulls.
Feeding linseed decreased (P < 0.05) the expression
of stearoyl CoA desaturase (SCD) and the lipoprotein
lipase (LPL) gene without affecting (P ≥ 0.19) fatty acid
synthase (FASN), leptin (LEP), and PPARγ2 mRNA
in the s.c. fat of bulls; however, there was no effect of
bull breed (P ≥ 0.11) or interactive effect of breed and
linseed (P ≥ 0.23) on gene expression. Expression of
PPARγ2 was positively correlated with SCD (r = 0.454;
P = 0.01), LEP (r = 0.500; P < 0.01), and LPL (r = 0.531;
P < 0.01) mRNA, indicating that PPARγ2 increases the
expression of genes involved in lipogenesis.
ABSTRACT: The objective of this trial was to determine
the effect of breed and long-term dietary linseed
addition on composition of fatty acids and expression
of some genes involved in the lipid metabolism within
subcutaneous (s.c.) adipose tissue of young bulls. Italian
Simmental and Holstein bulls (n = 16/breed) were fed
a corn silage–grass hay diet with or without 8% (DM
basis) whole ground linseed. Inclusion of linseed, rich
in α-linoleic acid (C18:3n-3), increased (P < 0.05) the
proportions of linolelaidic (C18:2n-6trans), γ-linolenic
(C18:3n-6), C18:3n-3, and rumenic (cis9,trans11
conjugated linoleic acid) acids, as well as total n-3
fatty acid, total PUFA, and PUFA:SFA, but decreased
(P < 0.05) weight percentages of myristic (C14:0),
pentadecanoic (C15:0), palmitic (C16:0), palmitelaidic
(C16:1n-9trans), and margaric (C17:0) acids, along with
n-6:n-3, in the s.c. fat of young bulls. Even though PUFA
Key words: adipose tissue, beef cattle, breed, fatty acid, gene expression, linseed
© 2013 American Society of Animal Science. All rights reserved.
INTRODUCTION
It is widely recognized that unsaturated fatty acids
(UFA), in particular the n-3 PUFA, have beneficial
effects on human health. It has been reported that the
incidence of cardiovascular disease can be reduced by
replacing SFA with PUFA in the human diet (EFSA
Panel on Dietetic Products, Nutrition, and Allergies,
2010). There are 3 major factors influencing the fatty
acid composition of beef: 1) diet, 2) breed type, and 3)
1This study was funded by the Agency for Rural Development of
the Friuli Venezia Giulia Region (ERSA). The technical support of E.
Bianco, D. Davanzo, E. Simonetti (ERSA), and the staff of Marianis
Farm (ERSAGRICOLA) was greatly appreciated.
2Corresponding author: [email protected]
Received December 22, 2011.
Accepted September 6, 2012.
405
J. Anim. Sci. 2013.91:405–412
doi:10.2527/jas2011-5057
age of animal (Smith et al., 2009a). Furthermore, the
fatty acid composition of adipose tissue can be derived
from intracellular de novo synthesis and lipoprotein
triacylglycerols (Cryer, 1985), and subcutaneous (s.c.)
adipose tissue is the principle site of lipogenesis in
cattle (Herdmann et al., 2010).
The effect of linseed supplementation on fatty
acid composition is well documented and is often
accompanied by an increase in linolenic acid (C18:3n-3)
and n-3 long-chain fatty acids in tissue (Scollan et al.,
2006). Conversely, breed has only minor effects on
fatty acid composition of cattle (De Smet et al., 2004).
Several lipogenic genes are regulated by genetic
and nutritional factors (Hocquette et al., 2007), but
relatively little information concerning the effect of
dietary lipid supplementation (especially linseed) on the
expression of genes involved in the lipid metabolism is
406
Corazzin et al.
available in ruminants (Chen et al., 2010; Herdmann et
al., 2010), and the metabolic origins of the differences
between breeds are poorly understood (Bonnet et al.,
2007). It was hypothesized that differences in fatty acid
composition between breeds may have genetic origins
and that linseed can influence the fatty acid profile of
young bulls by providing increased quantities of linolenic
acid and affecting the expression of specific genes. Thus,
the objective of this trial was to determine the effect of
whole ground linseed supplementation on the expression
of some lipid metabolism genes in the adipose tissue of
Italian Simmental and Holstein young bulls.
MATERIALS AND METHODS
All procedures meet the requirements of European
Community Directive 86-609-EC for Scientific
Procedure Establishments.
Experimental Design and Sample Collection
At approximately 5 mo of age, weaned Italian
Simmental (IS) and Italian Holstein (IH) bulls, with an
initial BW of 171 ± 7.3 and 181 ± 5.4 kg, respectively,
were fed corn silage–grass hay–based diets with (WL)
or without (CON) 8% (DM basis) whole ground linseed.
Pens of bulls (8 bulls/pen) were offered a growth diet for
the first 90 d, after which they were adapted to a finishing
diet (Table 1). Diets were isocaloric and isonitrogenous
and totally mixed and were provided once daily in the
morning. Holstein and Simmental bulls were transported
15 km and slaughtered at an average BW of 577 ± 24.5 kg
(496 ± 19.3 d of age) and 619 ± 19.3 kg (476 ± 24.6 d
of age), respectively, at an EU-licensed abattoir within
30 min of arrival. Approximately 10 g of s.c. fat from the
tail head were obtained within 20 min of exsanguination,
frozen in N2, and stored at −80°C until analysis.
Fatty Acids Analysis
Extraction of total lipids was performed according to
the procedure of Folch et al. (1957). Fifteen milligrams
of nonadecanoic acid (C19:0) were added to 1.5 g of
minced s.c. fat and homogenized in 30 mL of chloroformmethanol mixture (2:1 vol/vol) using an Ultra-Turrax
homogenizer (T 25 basic; Ika-Werke, Staufen, Germany)
and were subsequently filtered under vacuum through
Whatman 1820-047 filter paper (GE Healthcare, Little
Chalfont, UK). The extract was washed with 8.5 mL of
0.88% (wt/vol) KCl, mixed vigorously for 1 min, and then
left overnight at room temperature. The organic phase
was separated, and the solvents were evaporated under
vacuum at 40°C. Fatty acid methyl esters (FAME) were
prepared using HCl methanolic (Sukhija and Palmquist,
1988). Lipid samples were mixed with 2 mL of hexane
and 3 mL of HCl methanolic in 20-mL glass tubes with
Teflon-lined caps. The mixture was heated at 70°C for 2
h, then cooled to room temperature before FAME were
extracted in 2 mL of hexane after addition of 5 mL of 6%
(wt/vol) K2CO3 and Na2SO4. Samples stood for 30 min
before centrifugation at 1,006 × g for 10 min at 20°C,
and the upper hexane layer was removed, concentrated
under N2, and then diluted in hexane. The FAME were
quantified using a Carlo Erba gas chromatograph (GC;
HRGC 5300 mega-series; Rodano, Milan, Italy) fitted
with an automatic sampler (model A200S; Rodano,
Milan, Italy) and a flame ionization detector (FID),
where 1 μL of sample was injected in 1:30 split mode.
The GC was equipped with a 60-m SP-2380 fused
silica capillary column (0.25 mm i.d., film thickness
of 0.25 μm; Supelco, Inc., Bellefonte, PA), and oven
temperature increased from 160°C to 180°C at 1°C/min,
then from 180°C to 260°C at 5°C/min, and then held for
5 min. Helium was the carrier gas at the rate of 1.2 mL/
min, and FAME were identified using external standards
(Supelco 37 components FAME mix, linoleic acids
conjugated; Sigma-Aldrich, Milan, Italy), quantified
using C19:0 as the internal standard and expressed as
percentage of the total lipids identified.
Table 1. Ingredients and chemical composition of diet
Growing
Finishing
Item
Control Linseed Control Linseed
Ingredients, % of DM
Maize silage
18.4
18.2
18.1
18.8
Grass hay
25.1
24.8
24.7
25.7
Wheat straw
6.0
6.0
5.9
6.2
Maize meal
25.8
22.9
28.3
20.0
Barley meal
5.7
4.3
5.7
4.6
Whole soybean
17.5
17.4
14.7
14.4
Whole linseed
0.0
5.0
0.0
8.0
Mineral-vitamin premix1
1.5
1.4
2.7
2.5
Chemical composition
CP, % of DM
14.3
14.7
13.5
14.0
Crude fiber, % of DM
18.2
17.6
16.4
18.2
Ash, % of DM
8.2
8.2
8.1
8.2
NEF,2 MJ/kg DM
7.23
7.30
7.21
7.22
Fatty acid composition, g/kg DM
C14:0
0.1
0.1
0.1
0.1
C16:0
7.8
8.7
7.7
8.7
C18:0
2.0
2.5
1.9
2.6
C18:1n-9
12.9
15.2
12.3
15.5
C18:2n-6
22.9
25.3
21.8
24.6
C18:3n-3
3.6
7.6
3.3
10.3
Total fatty acid, g/kg DM
50.5
60.0
48.0
63.2
1Provided 150 g Ca, 50 g P, 80 g Na, 32 g Mg, 1.2 g Fe, 3.5 g Zn, 1.2g
Mn, 0.2 g Cu, 25 mg Co, 35 mg I, 1 mg Se, 500,000 IU vitamin A, 50,000 IU
vitamin D3, 1,000 mg vitamin E, 400 mg vitamin B1, and 2,000 mg vitamin
B3 per kg of diet.
2NEF = net energy for fattening, calculated according to Institut National
de la Recherche Agronomique (INRA) standards (Vermorel, 1988).
407
Linseed and adipose gene expression
RNA Extraction and cDNA Synthesis
Total cellular RNA was extracted from 40 mg
of s.c. adipose tissue using a RNeasy Lipid Tissue
Mini Kit (Qiagen, Hilden, Germany) according to the
manufacturer’s protocol. Concentration and integrity
of total RNA were determined and checked using the
Agilent 2100 bioanalyzer (Agilent Technologies, Palo
Alto, CA). All s.c. fat samples had an RNA integrity
number (RIN) greater than 7. To obtain cDNA, an iScript
cDNA Synthesis kit (BioRad, Milan, Italy) was used.
Each 21 μL of reaction contained 15 μL of RNA, 1 μL
of RNase H+ Moloney murine leukemia virus (MMLV)
reverse transcriptase (200 U/μL), 4 μL of 5X iScript
reaction mix, composed of 1 μL of deoxynucleoside
triphosphate (dNTP, 10 mM), 2 μL of dithiothreitol
(DTT; 0.1 M), 4 μL of 5X Buffer [250 mM Tris-HCl
(pH 8.3), 375 mM KCl, and 15 mM MgCl2], and 1 μL
of nuclease-free water. The mixture was held for 5 min
at 25°C, 1 h at 42°C, and 5 min at 85°C before being
cooled to 16°C. The cDNA was quantified using a
spectrophotometer (TM 2000; Nano-Drop Technologies,
Rockland, DE) and stored at −20°C.
Real-Time (RT) PCR Analysis
A qualitative PCR was carried out with the objective
of verifying the primers (primer sequences are presented
in Table 2). In particular, 1 μL of cDNA was added to a
mixture containing 0.20 μL of each forward and reverse
primer (20 μM), 8.6 μL of sterile water, and 10 μL of
iQ SYBR Green Supermix [100 mM KCl, 40 mM TrisHCl (pH 8.4), 0.4 mM of dNTP, 50 U/mL of iTaq DNA
polymerase, 6 mM MgCl2, SYBR Green I, and 20 nM
fluorescein; BioRad]. Amplification conditions were 1
cycle of 3 min at 95°C, followed by 39 PCR cycles of
15 s at 95°C, 30 s at the annealing temperature of the
primers (60°C), 30 s at 72°C, and a final extension for 10
min at 72°C. The amplified PCR products were examined
by electrophoresis in 1.5% (wt/vol) agarose gel.
The quantitative PCR (qPCR) was performed using
the BioRad CFX96 system (BioRad, Hercules, CA) on
a reaction volume of 20 μL, containing 0.20 μL of each
forward and reverse primer (20 μM), 10 μL of iQ SYBR
Green Supermix (BioRad), 8.6 μL of sterile water, and
1 μL of cDNA. The amplification conditions included 1
cycle of 3 min at 95°C, 39 PCR cycles of 15 s at 95°C,
30 s at 60°C, and 30 s at 72°C, followed by a dissociation
step of 5 min at 70°C, 5 s at 95°C, 30 s at 55°C, 5 s at
65°C, and 5 s at 90°C.
Each sample was analyzed in triplicate, and the
average was used to calculate the relative gene expression
according to the efficiency-corrected method proposed
by Pfaffl (2001). In particular, β-actin, cyclophilin,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
and ribosomial protein large P0 (RPLP0) were treated
as reference genes. For the normalization of the RTqPCR data, only GAPDH, RPLP0, and cyclophilin were
considered because they presented better average genestability measure (M < 0.15) after geNorm analysis, and
their geometric mean was used (Vandesompele et al.,
Table 2. Primer sequences (5′ to 3′) and real-time amplification products
Gene
LEP1
SCD2
LPL3
FASN4
PPARγ2
Cyclophilin
β-actin
GAPDH5
RPLP06
1LEP =
Primers, Forward-Reverse
F: TCTGTCTTACGTGGAGGCTGTGC
R: TCTGTTTGGAGGAGACGGACTGC
F: TTATTCCGTTATGCCCTTGG
R: GGTAGTTGTGGAAGCCCTCA
F: TTCAGAGGCTATTACTGGAAATCC
R: ATGTCAATCACAGCATTCATTCTACT
F: ACAGCCTCTTCCTGTTTGACG
R: CTCTGCACGATCAGCTCGAC
F: AGGATGGGGTCCTCATATCC
R: GCGTTGAACTTCACAGCAAA
F: GGATTTATGTGCCAGGGTGGTGA
R: CAAGATGCCAGGACCTGTATG
F: CTCTTCCAGCCTTCCTTCCT
R: GGGCAGTGATCTCTTTCTGC
F: TCATCCCTGCTTCTACTGGC
R: CCTGCTTCACCACCTTCTTG
F: CAACCCTGAAGTGCTTGACAT
R: AGGCAGATGGATCAGCCA
leptin
= stearoyl CoA desaturase.
3LPL = lipoprotein lipase.
4FASN = fatty acid synthase.
5GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
6RPLP0 = ribosomial protein large P0
2SCD
Amplicon (bp)
122
Accession number or reference
U43943
R2
0.995
Efficiency
0.99
150
Duckett et al. (2009)
0.999
0.98
186
Bernard et al. (2005)
0.998
0.88
226
Bernard et al. (2005)
0.997
0.95
120
Duckett et al. (2009)
0.997
0.95
119
AY247029
0.997
0.93
177
Duckett et al. (2009)
0.999
0.91
177
U85042
0.996
1.01
226
Wang et al. (2009)
0.998
0.92
408
Corazzin et al.
2002). The data were presented in a fold-change ratio
having as a reference the IS CON group, and primer
efficiency was calculated and verified using the standard
curve obtained by serial dilution of the pooled cDNA
(Pfaffl, 2001).
Statistical Analysis
Normality of data distribution was tested with the
Kolmogorov-Smirnov test, and where appropriate,
nonparametrically distributed data were transformed for
parametric testing. The ANOVA was generated using
SPSS (SPSS Inc., Chicago, IL) to determine the main
effects of diet, breed, and diet × breed interaction on
fatty acid composition and expression of stearolyl CoA
desaturase (SCD), fatty acid synthase (FASN), PPARγ2,
and the lipoprotein lipase (LPL) gene. In the same
model, carcass fatness (estimated by dissection of the
eighth rib sample joint; Andrighetto et al., 1996) was
included as a covariate for leptin (LEP) gene expression
analysis. Rho Spearman coefficients were used to
determine associations between variables. A probability
of P ≤ 0.05 was established for statistical significance.
RESULTS AND DISCUSSION
The proportion PUFA did not (P ≥ 0.23) differ
between breeds of bulls, but s.c. fat from Holstein bulls
had greater (P < 0.05) proportions of tridecylic (C13:0),
myristoleic (C14:1), and palmitoleic (C16:1n-9cis) acids
and a decreased (P < 0.05) proportion of margaric acid
(C17:0) than s.c. fat from Simmental bulls (Table 3).
These results are in agreement with Siebert et al. (1996),
who found only minor differences between breeds in
the fatty acid composition of s.c. fat. Choi et al. (2000)
reported differences in the s.c. fat content of pentadecanoic
(C15:0), C17:0, stearic (C18:0), C18:1trans fatty acid, cisvaccenic (C18:1n-7), and oleic (C18:1n-9) acids between
Holstein Friesian and Welsh Blacks steers slaughtered at
different fatness scores.
Proportions of myristic (C14:0), C15:0, and palmitic
(C16:0) acids were lower (P < 0.05) in bulls fed whole
ground linseed (Table 3). These results are consistent
with the findings of Scollan et al. (2001), who reported
a decrease in the proportions of C16:0 in s.c. fat of beef
cattle fed whole linseed. Additionally, Mach et al. (2006)
reported that feeding bulls diets rich in UFA reduced
de novo fatty acid synthesis in the rumen and adipose
tissue. Also, the proportions of C:17:0 and palmitelaidic
(C16:1n-9trans) acids were less (P < 0.05) in bulls fed
whole ground linseed, but feeding linseed increased (P <
0.01) the proportion of linolelaidic (C18:2n-6trans),
γ-linolenic (C18:3 n-6), and rumenic (cis-9,trans-11
CLA) acids. These results are in agreement with the study
of Bartoň et al. (2007), who reported that feeding linseed
increased the proportion of CLA and had only a marginal
effect on the desaturase index in s.c. adipose tissue. The
interaction between experimental factors was significant
for the proportion of total n-3 fatty acids, which was
largely a response to greater (P < 0.01) deposition of
C18:3n-3. In agreement with other studies (Raes et
al., 2004; Herdmann et al., 2010), bulls fed 8% whole
ground linseed had greater (P < 0.01) proportions of total
n-3 fatty acids, with a greater effect in Simmental than
Holstein bulls (P < 0.05). Feeding whole ground linseed
reduced (P < 0.01) the n-6:n-3, but weight percentages of
total PUFA and the PUFA:SFA were greater (P < 0.05) in
linseed-fed bulls.
The expression of the LEP gene was not (P = 0.37)
affected by breed of bull (Table 4). Leptin is a protein
hormone synthesized and expressed predominantly in the
adipose tissue, and it plays a key role in the regulation of
appetite, energy partition, and body composition (Liefers
et al., 2002). Moreover, LEP gene expression is modulated
according to different physiological states of the animal
and seems to be stimulated by increased fat deposition
(Agarwal et al., 2009). In the present trial, the adjustment
of LEP mRNA abundance with carcasses fat could explain
the lack of differences in LEP gene expression between
breeds. Chilliard et al. (2005) reported that the LEP
mRNA abundance was lower in leaner breeds of cattle;
however, these differences disappeared when breeds were
compared at similar adipocyte size.
Dietary linseed supplementation did not (P = 0.61)
affect LEP gene expression (Table 4). The effect of
diet on LEP gene expression is not fully understood.
In general, UFA reduces LEP expression in bovine
adipocytes by mechanisms that involve PPARγ gene
expression (Reseland et al., 2001). The results of the
present study are in agreement with the findings of
Bonnet et al. (2009), who noted that feeding linseed did
not modify the LEP mRNA abundance in adipose tissue
of goats, highlighting the lack of an effect of α-linolenic
acid (C18:3n-3) and its biohydrogenation products. The
same authors suggested that the production of C18:1trans
isomers in the rumen could be involved in the regulation
of LEP gene expression. However, in the present trial,
the proportion of C18:1trans isomers was not (P = 0.67)
affected by feeding 8% whole ground linseed.
Stearoyl CoA desaturase is an enzyme involved
in the conversion of SFA into MUFA by introducing
a double bond at the ∆9 position of the fatty acid. In
the current study, breed did not (P = 0.41) affect
SCD gene expression (Table 4). The molecular basis
that underscores any differences in SCD expression
among breeds is still not clear (Ohsaki et al., 2007),
but Taniguchi et al. (2004) reported greater SCD gene
expression and MUFA content in s.c. fat of Japanese
Linseed and adipose gene expression
409
Table 3. Fatty acid composition (weight percentage) of subcutaneous fat of Italian Simmental (IS) and Italian Holstein
(IH) bulls fed a diet without (CON) or with 8% whole ground linseed (WL)
IS
IH
P-value1
WL
CON
WL
CON
SEM
B
D
B×D
8
8
8
8
0.02
0.02
0.02
0.02
0.001
0.17
0.20
0.08
0.03
0.03
0.03
0.03
0.001
0.74
0.09
0.81
0.09
0.11
0.09
0.12
0.005
0.81
0.09
0.46
0.12
0.11
0.15
0.14
0.006
0.01
0.55
0.97
2.96
3.40
3.11
3.42
0.080
0.58
0.02
0.68
0.48
0.55
0.57
0.75
0.036
0.04
0.07
0.39
0.36
0.45
0.38
0.44
0.013
0.77
<0.01
0.58
0.54
0.54
0.61
0.60
0.027
0.26
0.91
0.95
23.89
25.38
23.31
24.78
0.223
0.11
<0.01
0.98
0.36
0.44
0.36
0.42
0.014
0.73
0.01
0.74
Palmitelaidic acid (C16:1n-9trans)
2.41
2.19
2.72
2.88
0.110
0.02
0.89
0.36
Palmitoleic acid (C16:1n-9cis)
Margaric acid (C17:0)
0.83
0.92
0.77
0.84
0.019
0.04
0.02
0.73
Heptadecenoic acid (C17:1)
0.49
0.46
0.50
0.53
0.014
0.14
0.98
0.25
Stearic acid (C18:0)
21.70
22.13
22.75
19.90
0.653
0.66
0.85
0.22
2.25
2.09
1.91
2.14
0.051
0.15
0.67
0.06
trans-Oleic acid (C18:1trans)2
34.88
33.26
34.47
35.04
0.659
0.62
0.70
0.43
Oleic acid (C18:1n-9cis)
2.41
2.21
2.24
2.18
0.039
0.19
0.10
0.38
cis-Vaccenic acid (C18:1n-7)
0.40
0.36
0.46
0.32
0.017
0.65
<0.01
0.08
Linolelaidic acid (C18:2n-6trans)
4.30
4.38
4.19
4.44
0.068
0.86
0.25
0.55
Linoleic acid (C18:2n-6cis)
γ-Linolenic acid (C18:3n-6)
0.16
0.07
0.16
0.07
0.010
0.68
<0.01
0.57
α-Linolenic acid (C18:3n-3)
0.94a
0.50c
0.79b
0.56c
0.039
0.27
<0.01
0.03
0.11
0.11
0.14
0.10
0.005
0.31
0.02
0.06
Rumenic acid (cis9,trans11 CLA)
0.05
0.05
0.05
0.05
0.002
0.70
0.87
0.39
CLA trans10, cis12
Eicosenoic acid (C20:1n-9)
0.15
0.15
0.16
0.13
0.006
0.68
0.18
0.09
Eicosadienoic acid (C20:2n-6)
0.03
0.04
0.04
0.04
0.003
0.97
0.40
0.37
Arachidonic acid (C20:4n-6)
0.04
0.05
0.05
0.05
0.003
0.23
0.43
0.38
SFA3
50.00
52.54
50.60
49.70
0.728
0.45
0.58
0.25
MUFA4
43.97
41.89
43.53
44.68
0.749
0.45
0.77
0.30
Total n-6 PUFA5
4.93
4.90
4.89
4.92
0.067
0.95
0.98
0.85
Total n-3 PUFA6
0.94a
0.50c
0.79b
0.56c
0.039
0.27
<0.01
0.03
PUFA7
6.03
5.56
5.87
5.62
0.088
0.76
0.04
0.52
MUFA:SFA
0.900
0.803
0.869
0.908
0.027
0.50
0.60
0.22
PUFA:SFA
0.122
0.106
0.116
0.113
0.002
0.86
0.03
0.14
n-6:n-3
5.26
10.25
6.82
8.90
0.498
0.89
<0.01
0.07
∆9 desaturase index8
45.20
42.82
44.71
45.71
0.806
0.47
0.68
0.31
a–cWithin a row, means without a common superscript letter differ (P < 0.05).
1B = breed and D = diet.
2trans-Oleic acid = C18:1n-11trans + C18:1n-9trans.
3SFA = C8:0 + C10:0 + C12:0 + C13:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0.
4MUFA = C14:1 + C15:1 + C16:1n-9trans + C16:1n-9cis + C17:1 + C18:1trans + C18:1n-9cis + C18:1n-7 + C20:1n-9.
5PUFA n-6 = C18:2n-6trans + C18:2n-6cis + C18:3n-6 + C20:2n-6 + C20:4n-6.
6PUFA n-3 = C18:3n-3.
7PUFA = PUFA n-3 + PUFA n-6.
8∆9 desaturase index = (C16:1 + C18:1n-9cis + C18:1n-7)/(C16:1 + C18:1n-9cis + C18:1n-7 + C14:0 + C16:0 + C18:0) × 100 (Smith et al., 2002).
Fatty
acid
Bull numbers
Caprylic acid (C8:0)
Capric acid (C10:0)
Lauric acid (C12:0)
Tridecylic acid (C13:0)
Myristic acid (C14:0)
Myristoleic acid (C14:1)
Pentadecanoic acid (C15:0)
Pentadecenoic acid (C15:1)
Palmitic acid (C16:0)
Black bulls than those of the Holstein breed. Smith et
al. (2009b) suggested that SCD gene expression was
associated with greater accumulation of MUFA in
adipose tissue of cattle, and the lack of a difference in
SCD gene expression between breeds appears to mirror
the lack of a breed effect on MUFA content (Table 3).
Despite similar MUFA percentages (P = 0.77) and
∆9 desaturase index (P = 0.68) between diets (Table 3),
SCD gene expression was reduced (P < 0.05) by feeding
whole ground linseed. Surprisingly, there was no (P >
0.05) correlation between ∆9 desaturation index and SCD
gene expression (results not shown). Waters et al. (2009)
demonstrated that SCD gene expression in muscle could
410
Corazzin et al.
be reduced by PUFA n-3-enriched diets, and Herdmann
et al. (2010) revealed that diets rich in n-3 fatty acid were
able to reduce SCD protein expression in s.c. fat of bulls
without altering the MUFA composition. Also, Dervishi
et al. (2010) reported that SCD gene expression was
reduced in the muscle of lambs in the presence of high
levels of CLA and low n-6:n-3. Moreover, Archibeque et
al. (2005) argued that the ∆9 desaturase index does not
correctly reflect the enzyme activity in cattle.
Expression of the LPL gene was not (P = 0.52)
affected by bull breed (Table 4). Lipoprotein lipase is
the rate-limiting enzyme that hydrolyzes triglycerides
from chylomicrons and very low density lipoprotein
(VLDL), providing FFA and 2-monoacylglycerol for
tissue utilization (Cryer, 1981). Furthermore, LPL plays
a key role in the control of triacylglycerol partitioning
between muscles and adipose depots (Hocquette et al.,
1998); therefore, expression of the LPL gene depends on
the physiological state and energy balance of cattle. In
the present trial, the same caloric density of the treatment
diet could explain the lack of a breed effect on LPL
mRNA abundance in s.c. fat. Ren et al. (2002) showed
that LPL mRNA abundances were similar between the
s.c. fat depots of German Holstein and Charolais bulls
fed similar nutritional levels but slaughtered at different
body fat contents.
Adding 8% linseed to experimental diets reduced
(P < 0.05) LPL gene expression (Table 4). Waylan
et al. (2004) demonstrated that feeding steers a diet
supplemented with linseed for 28 d decreased the
LPL mRNA abundance in muscle tissue. Moreover,
Hocquette et al. (2001) hypothesized that the decrease in
absorbed fat could decrease LPL gene expression; thus,
it is plausible that the different fat levels of experimental
diets could have affected the expression of this gene. In
the current trial, LPL mRNA abundance was positively
correlated with oleic acid (r = 0.481; P < 0.01), total
MUFA (r = 0.491; P < 0.01), and MUFA:SFA (r = 0.482;
P < 0.01) but negatively correlated with total SFA (r =
−0.459; P < 0.01; results not shown). These results could
be explained because the LPL has a positional specificity
for the fatty acid release (18:1 > 18:3 > 18:2 > 14:0 >
16:0 > 18:0; Wang et al., 1982).
Fatty acid synthase mRNA abundance was similar
(P = 0.56) between Simmental and Holstein bulls
(Table 4). Fatty acid synthase is a multifunctional
enzyme that catalyzes the last step in the fatty acid
biosynthetic pathway producing C16:0 from acetyl-CoA
and malonyl-CoA (Ordovás et al., 2008). Pickworth et
al. (2011) reported a positive correlation between FASN
mRNA abundances and external fat thickness of AngusSimmental crossbred steers. Therefore, the similar FASN
mRNA abundances between breeds could be due to the
decision to slaughter the bulls at a commercial target
weight that corresponded to similar carcass fat scores.
Fatty acid synthase expression did not (P = 0.19)
statistically differ between diets (Table 4). However, a
numerical reduction in FASN expression was observed and,
together with the reduced proportions of C14 (P = 0.02)
and C16:0 (P < 0.01), could be an indication that whole
ground linseed reduces FASN mRNA abundance. Hiller et
al. (2011) showed that increasing n-3 PUFA composition
in the diet of Holstein bulls caused a significant reduction
in FASN gene expression in s.c. fat. Even though FASN
is widely considered a key enzyme in de novo fatty acid
synthesis, there were no (P > 0.05) correlations between
FASN mRNA expression and proportions of C14:0, C16:0,
and total SFA (results not shown).
Abundance of PPARγ2 mRNA was similar (P =
0.11) between breeds (Table 4). Peroxisome proliferatoractivated receptor γ is a member of a subfamily of
ligand-activated nuclear receptors that includes PPARα
and PPARβ/δ (Fernyhough et al., 2007), and it plays a
key role in adipocyte differentiation, fatty acid uptake,
de novo fatty acid synthesis, and lipolysis and is the
major regulator of lipid storage in the adipose tissue by
modulating the expression of several genes involved in
fatty acid metabolism (Feige et al., 2006). Results of the
present study concur with those of Huff et al. (2004),
who did not find differences in PPARγ gene expression
between Charolais and Holstein cattle slaughtered at
the same external fat thickness. Additionally, PPARγ2
is involved in adipose cells differentiation (Fernyhough
et al., 2007), but Joseph et al. (2010) suggested that the
majority of adipocyte differentiation occurs within 8 mo
of age in beef cattle; thus, it is plausible that the lack
of an effect of bull breed on PPARγ2 expression was
because bulls were greater than 8 mo of age at slaughter
and the adipocyte differentiation was possibly complete.
Table 4. Fold change ratio in relative RNA expression
of genes of subcutaneous fat of Italian Simmental (IS)
and Italian Holstein (IH) bulls fed a diet without (CON)
or with 8% whole ground linseed (WL); groups were
compared with the IS CON group (Pfaffl, 2001; n = 32)
IS
IH
Gene
WL
CON
WL
CON
LEP2
1.17
1.00
0.44
0.98
SCD3
0.45
1.00
0.38
0.97
LPL4
0.59
1.00
0.56
0.84
FASN5 0.50
1.00
0.53
0.82
PPARγ2 0.87
1.00
0.82
0.73
1B = breed and D = diet.
2LEP = leptin.
3SCD = stearoyl CoA desaturase.
4LPL = lipoprotein lipase.
5FASN = fatty acid synthase.
SEM
0.378
0.476
0.333
0.428
0.286
B
0.37
0.41
0.52
0.56
0.11
P-value1
D
0.61
0.04
0.04
0.19
0.32
B×D
0.74
0.99
0.23
0.49
0.49
Linseed and adipose gene expression
Feeding linseed did not (P = 0.32) influence the
PPARγ2 expression (Table 4). Many UFA are activators
of PPARγ (Fernyhough et al., 2007), and Clarke (2000)
observed that PPARγ mRNA abundance was more
influenced by n-6 and n-3 PUFA. Moreover, PPARγ2
gene expression was positively correlated with SCD (r =
0.454; P = 0.01), LEP (r = 0.500; P < 0.01), and LPL
(r = 0.531; P < 0.01) but not (r = 0.330; P = 0.07) with
FASN mRNA abundance (results not shown). These
positive correlations indicate that PPARγ2 increases
the expression of genes involved in lipogenesis. Also,
Dervishi et al. (2010) found a correlation between
PPARγ and SCD gene expression (r = 0.554) without
any dietary effect on PPARγ.
In summary, differences in fatty acid composition
between breeds were minor, and breed did not affect the
expression of genes involved in the lipid metabolism. On
the other hand, feeding diets containing up to 8% whole
ground linseed increased the percentage of C18:2n6trans, C18:3n-3, C18:3n-6, cis9,trans11 CLA, total
n-3 PUFA, total PUFA, and PUFA:SFA but decreased
the proportions of C14:0, C15:0, C16:0, C16:1n-9trans,
C17:0, and n-6:n-3 in s.c. adipose tissue of young bulls.
Furthermore, the inclusion of linseed in diets of young
bulls decreased the expression of SCD and LPL genes,
without affecting the expression of FASN, LEP, and
PPARγ2 gene in adipose tissue. However, the positive
correlations between the abundances of PPARγ2 and
SCD, LEP, and LPL mRNA indicated that PPARγ2
increases the expression of genes involved in lipogenesis.
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