J. Anim. Breed. Genet.
ISSN 0931-2668
ORIGINAL ARTICLE
Association of a melanocortin 4 receptor (MC4R) polymorphism
with performance traits in Lithuanian White pigs
R. Jokubka1, S. Maak2, S. Kerziene1 & H. H. Swalve2
1 Department of Animal Breeding and Genetics, Lithuanian Veterinary Academy, Kaunas, Lithuania
2 Institute of Animal Breeding and Husbandry with Veterinary Clinic, Martin-Luther-University Halle-Wittenberg, Halle, Germany
Correspondence
Ramunas Jokubka, Department of Animal
Breeding and Genetics, Lithuanian Veterinary
Academy, Tilzes 18, LT-47181 Kaunas,
Lithuania. E-mail: ramunas.jokubka@lva.lt
Received: 3 January 2005;
accepted: 19 September 2005
Introduction
The identification of genes affecting fatness, growth
rate and meat quality is of crucial interest for the
swine industry. However, only few candidates for
these quantitative traits have been reported so far.
The melanocortin 4 receptor (MC4R) gene codes for
a G-protein-coupled receptor and it was demonstra-
ted to be important in the control of energy balance
in humans and rodents (Seeley et al. 1997). Energy
balance is maintained by controlling energy intake,
i.e. feed intake, and energy expenditure by physical
activity and metabolism (Marie et al. 2000). The
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Journal compilation ª 2006 Blackwell Verlag, Berlin • J. Anim. Breed. Genet. 123 (2006) 17–22
Summary
The melanocortin 4 receptor is expressed in virtually all brain regions of
mammals and plays an important role in energy homeostasis. Polymor-
phisms in this gene may thus be related to growth and obesity. In pigs,
a non-synonymous polymorphic site was described (Asp298Asn) and
demonstrated to affect cAMP production and to alter adenylyl cyclase
signalling. Association studies revealed significant linkage of this muta-
tion with production trait in pigs. In this study, 207 Lithuanian White
pigs were genotyped at the MC4R locus and analysed on relationships
between genotype and breeding values for several performance traits.
The observed allele and genotype frequencies did not deviate signifi-
cantly from Hardy–Weinberg equilibrium (wildtype allele 0.59; mutant
allele 0.41) and are comparable with those described in other Large
White populations. The mutant Asn298 allele of the MC4R gene was
significantly associated with increased test daily gain, higher lean meat
percentage and lower backfat thickness. There was a trend towards an
improved feed conversion ratio (p ¼ 0.065) in animals with the mutant
allele whereas no significant effect was found on lifetime daily gain.
These results indicate that the MC4R polymorphism should be integra-
ted in selection programmes in the Lithuanian White to improve carcass
composition.
response of the melanocortin 4 receptor to leptin sig-
nalling can thus be considered as a link between
feed intake and body weight and body composition
(Marsh et al. 1999; Wikberg et al. 2000). Conse-
quently, MC4R is a strong candidate for growth and
body composition in pigs. Previous research on the
regulation of energy homeostasis showed the signifi-
cant effects of mutations in the MC4R receptor gene
on obesity in mice and humans (Vaisse et al. 1998;
Yeo et al. 1998; Marsh et al. 1999). In contrast,
blocking of MC4R by an antagonist was demonstra-
ted to have no effect on the regulation of feed intake
of pigs (Barb et al. 2004). The porcine MC4R locus
17
MC4R polymorphism and performance traits in pigs
Traits TDG FCR BF
TDG 0.583
0.024 )0.693
0.018 0.148
0.044
FCR 0.305
0.018 0.078
0.038
BF 0.163
0.012
LDG
Meat %
TDG, test daily gain (in g/day); FCR, feed conversion ratio (in kg/kg); BF, backfat at 10th rib (in
mm); LDG, lifetime daily gain (in g/day); Meat%, lean meat percentage.
was mapped to SSC1 q22–q27 and a missense muta-
tion (Asp298Asn) was identified by Kim et al.
(2000a). This polymorphism was shown to be signifi-
cantly associated with feed intake, fatness and
growth traits in commercial lines (Kim et al. 2000b;
Hernandez-Sanchez et al. 2003) as well as in experi-
mental populations (Liu et al. 2002). However, the
effects were not observed in all investigated lines
and could not be confirmed by analysis of a Large
White · Wild Boar intercross (Kim et al. 2000b; Park
et al. 2002). Functional studies on the MC4R gene
demonstrated a decreased cAMP production in cells
expressing the mutant protein (Kim et al. 2004). This
missense mutation could therefore exert a direct
effect on the investigated phenotypic traits rather
than being a marker for a linked gene.
The Lithuanian White as a major breed in pro-
grammes for the production of fattening pigs in
Lithuania is characterized by a high backfat thick-
ness compared with commercial breeds and lines
from Western Europe. This underlines the need for
intensive selection for leanness that could be boosted
by marker-assisted selection. The objective of this
study was to evaluate the frequency of the
Asp298Asn mutation in the Lithuanian White popu-
lation and to investigate the association of this poly-
morphism with performance traits.
Materials and methods
Animals
Lithuanian White is used as a dam line in Lithuan-
ian breeding programmes. This line was bred by int-
rogression of British and Swedish Yorkshire and
German Edelschwein into Lithuanian indigenous
pigs during the early decades of the 20th century. It
was recognized as a separate breed in 1967 (Mako-
veckas 1986). The data set used for this study com-
prised 207 randomly chosen fattening pigs for
genotyping from a total of 16 539 Lithuanian White
pigs with performance records. Among the 207 gen-
otyped pigs, 111 and 96 had records from the test
18
R. Jokubka et al.
Table 1 Heritability coefficients (
SE; bold,
LDG Meat % diagonal) of the traits and genetic correla-
0.302
0.077 0.098
0.052 tions
SE (above diagonal) between the
)0.288
0.085 )0.172
0.026 traits estimated with VCE (Groeneveld 1998)
)0.147
0.041 )0.810
0.046 for the entire data set of performance
0.110
0.007 0.210
0.012 records (n ¼ 16 539)
0.292
0.012
station and on-farm tests respectively. The 207
experimental animals comprised full- and half-sib-
lings from 106 sows and 57 boars. These 57 boars
represent more than one-third of the total number
of boars used in this period in the Lithuanian White
population.
Fattening and performance traits
Fattening performance was tested in the weight
range between 30 and 100 kg at the State Pig Test-
ing Station. Fattening was performed under restric-
ted feeding conditions with maintenance of records
for each individual pig. The animals were slaugh-
tered at an average live weight of 100 kg and dis-
sected according to the station methodology. The
following traits were recorded: test daily gain (TDG)
calculated as total weight gain at the station divided
by the number of test days, feed conversion ratio
(FCR) as feed consumption per kilogram weight
gain in the test period, backfat at the 10th rib (BF)
determined by dissection (at station), lifetime daily
gain (LDG) calculated from weight on farm
(approximately 70–130 kg) divided by age at weigh-
ing (approximately 100–300 days), lean meat per-
centage (Meat%) at the end of the performance
test estimated with ultrasonic measurements PIG-
LOG 105 (SFK Technology, Søborg, Denmark) and
adjusted for live weight (at station and farm). The
age at measurements ranged from 5 to 7 months
with a weight range from 85 to 110 kg. To combine
both materials, breeding values were calculated
based on the parameters estimated for the total
population (Table 1).
Genotyping at the MC4R locus
Genomic DNA was extracted from hair roots accord-
ing to standard methods. The following primers were
designed with the software OLIGO 5.0 (MedProbe,
As, Norway) on the basis of the sequence published
by Kim et al. 2000a; GenBank entry AF087937):
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R. Jokubka et al.
5¢-ACA GTT AAG CGG GTT GGA AT-3¢ (forward)
and 5¢-CAG GGG ATA GCA ACA GAT GA-3¢
(reverse). A 483-bp fragment of the melanocortin 4
receptor (MC4R) gene was amplified in a total reac-
tion volume of 30 ll containing 10 ng genomic
DNA, 1.5 mm MgCl2, 0.2 mm of each dNTP, 1 lm of
each primer and 1 U Taq Polymerase (MBI Fermen-
tas, Vilnius, Lithuania). The PCR amplification was
performed in a GeneAmp
PCR System2700
(Applied Biosystem, Foster City, USA) with an initial
denaturation of 2 min at 94C followed by 35 cycles
with 30 s at 94C, 60 s at 58C, and 90 s at 72C. A
final elongation step (15 min at 72C) ended the
reaction. DNA fragments were digested with the
restriction endonuclease TaqI (MBI Fermentas; 2 U,
incubation for 1 h at 65C) and the resulting frag-
ments were separated by electrophoresis on 2% ag-
arose gels containing ethidium bromide. The
fragments were visualized and scored under ultravi-
olet light. The G/A polymorphism in the porcine
MC4R gene (Asp298Asn) resulted in 407- and 76-bp
fragments (wildtype: G nucleotide, Asp298), while
the absence of the TaqI cleavage site yielded a single
483-bp band (mutant: A nucleotide, Asn 298).
Statistical analysis
The multivariate animal model for genetic evalua-
tion of Lithuanian White pigs combined data from
progeny testing stations and breeding farms. The fol-
lowing traits were analysed: TDG, FCR, BF, LDG,
and Meat%. The breeding values were estimated
using the software packages PEST (Groeneveld 2001)
and VCE (Groeneveld 1998) applying the following
model for the entire data set of 16 539 pigs:
Yijlk ¼ l þ bo þ yshi þ sexj þ litterl þ animalk þ eijlk
with Yijlk the measured trait, l the population mean
of the trait, ysh the random joint effect of year, sea-
son and farm for traits measured on farm and the
fixed joint effect of station, year and season for traits
measured at station, ‘sex’ the fixed sex effect, ‘litter’
the random litter effect, ‘animal’ the additive-genetic
animal effect for all traits, b0 the regression of back
fat thickness on live weight, and e the residual error.
The chi-squared test was used to compare the
observed allele and genotype frequencies with
Hardy–Weinberg equilibrium assumption. The effect
of the bi-allelic MC4R polymorphism on the quanti-
tative performance traits was analysed with an one-
way anova approach using breeding values estimated
from the entire data set for genotyped animals. Post
hoc pairwise comparisons between genotypes were
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Journal compilation ª 2006 Blackwell Verlag, Berlin • J. Anim. Breed. Genet. 123 (2006) 17–22
MC4R polymorphism and performance traits in pigs
performed using Bonferroni adjustment. The homo-
geneity of the variances was evaluated using the Le-
vene test. To account for inhomogeneous variances,
a non-parametric Kruskal–Wallis rank-sum test was
used to analyse the effect of genotype on perform-
ance parameters. All statistical tests were carried out
using the SPSS (2003; version 12.0 for Windows).
Differences were considered to be significant if
p < 0.05.
Results and discussion
The estimated heritabilities for fattening traits
(Table 1) are in general accordance with results pub-
lished for Large White (Groeneveld et al. 1996; Nak-
avisut et al. 2003). However, the values for lean
meat percentage and backfat thickness are lower
than those described for various pig breeds (Peskovi-
cova et al. 2002). This may be attributed to different
methods applied in field test and testing station in
the investigated material. Noteworthy are the very
low standard errors for the estimations indicating a
high reliability of the resulting breeding values.
The observed genotype frequencies in our sample
were 0.343 and 0.169 for homozygous wildtype (GG)
and mutant animals (AA), respectively, and 0.488 for
heterozygotes (AG) resulting in allele frequencies of
0.59 for the wildtype and 0.41 for the mutant allele.
The population was tested to be free of the malignant
hyperthermia syndrome (MHS) thus excluding an
effect of this locus on the investigated traits. The vari-
ances of the traits BF and Meat% were not homogen-
eous in our sample, requiring the application of the
non-parametric Kruskal–Wallis test for the analysis of
the genotypic effects. The mean values of the popula-
tion as well as the deviation of the mean values of dif-
ferent MC4R genotypes for the analysed performance
traits are shown in Table 2. Single-marker analysis
revealed no significant association of MC4R gene with
FCR (p ¼ 0.058) and LDG (p ¼ 0.082); however, we
found statistically significant effects on TDG (p ¼
0.008), BF (p ¼ 0.043) and Meat% (p ¼ 0.030).
Homozygous mutant pigs had a significantly higher
TDG, a decreased BF and a higher Meat% than their
wildtype counterparts. FCR tended to be improved in
these pigs.
Previous investigations on a single-nucleotide
polymorphism in the porcine gene leading to an
Asp298Asn amino acid exchange revealed significant
relationships to traits related to obesity, growth and
feed intake (Kim et al. 2000b; Hernandez-Sanchez
et al. 2003; Houston et al. 2004). In contrast to many
other candidate genes (e.g. oestrogen receptor;
19
MC4R polymorphism and performance traits in pigs
MC4R genotype (nt298) (deviation of mean
SE)
Asp/Asp, Asp/Asn,
Trait lpop n ¼ 71 n ¼ 101
TDG (g) 729.1 )13.6
5.8 )13.5
4.8
FCR (kg/kg) 3.55 0.01
0.02 0.03
0.02
BF (mm) 22.5 )0.1
0.2 )0.2
0.2
LDG (g) 464.7 1.7
1.5 0.3
1.5
Meat (%) 53.2 0.1
0.2 0.2
0.2
p-values according to post hoc pairwise analysis (Bonferroni test) of the phenotypic variance.
Rothschild et al. 1996) which more likely serve as
markers for closely linked causal genetic variants
than bearing the causal mutation itself, the MC4R
receptor was demonstrated to act directly in the con-
trol of body weight and composition via mediating
the effect of leptin (Wikberg et al. 2000; Kim et al.
2004). The distribution of allele and genotype fre-
quencies for Asp298 and Asn298 in our sample did
not deviate significantly from Hardy–Weinberg equi-
librium (v2 ¼ 0.008, p ¼ 0.996, d.f. ¼ 2) indicating
that the locus is not under selection pressure. This
result (wildtype: 0.59 versus mutant: 0.41) is in the
same range as described for Large White-derived
lines (0.56 versus 0.44; Kim et al. 2000a). In con-
trast, the frequencies of this mutation were reported
to be 0.96 versus 0.04 and 0.90 versus 0.10 in two
Portuguese breeds which are characterized by a high
BF (Ramos et al. 2003). Houston et al. (2004)
observed a significantly shifted allele frequency
towards wildtype in a line selected for high lean feed
conversion; however, no shift in allele frequency
was observed in a line selected for high lean growth
under restricted feeding regimen which was dis-
cussed as a possible masking effect of genetic drift.
Daily weight gain at test was higher for the mutant
pigs and thus confirms the positive effect of the
Asp298Asn mutation on growth rate (Kim et al.
2000b; Hernandez-Sanchez et al. 2003; Houston et al.
2004). The direction of the reported allelic effects on
other traits, however, differed among the studies.
Kim et al. (2000b) and Houston et al. (2004) found
an increased backfat thickness being linked to the
mutant allele, whereas Park et al. (2002) reported a
non-significant opposite effect. Consideration of the
population stratification in the material used by Kim
et al. (2000b) and integration of additional data (Her-
nandez-Sanchez et al. 2003) seemed not to confirm
the decrease in BF in the homozygous mutant pigs,
thus apparently reversing the results of the initial
study. However, in this publication inadvertently a
different code was used for the genotypes (e.g. ‘11’
20
R. Jokubka et al.
Table 2 Population means and deviation of
means within MC4R genotypes for the traits
Asn/Asn, analysed (estimated breeding values)
n ¼ 35 p-value
8.7
5.6 0.046
)0.04
0.02 0.065
)1.0
0.3 0.020
4.2
2.0 0.445
1.0
0.3 0.007
for homozygous mutant instead for wildtype as used
in Kim et al. 2000b) so that the results finally con-
firm the first study (C.S. Haley, personal communi-
cation). In our study, using breeding values for the
traits, a significant decrease of BF in the order Asp/
Asp > Asp/Asn > Asn/Asn was observed. This
decrease was accompanied by a significant increase
of Meat% and a non-significant trend towards an
improved FCR. The allelic effects of the porcine
MC4R gene in Lithuanian White are consistent with
previous studies in general, except for fat deposition.
This may be due to the different feeding conditions
[restricted (this study) versus ad libitum (Kim et al.
2000b)] and to the limited number of animals inclu-
ded in this work. Under restricted feeding conditions,
the mutant Asn298 allele seems to shift energy util-
ization towards increased muscle weight gain and
decreased fatness. Kim et al. (2004) demonstrated a
functional effect of the Asp298Asn mutation leading
to a decreased cAMP production in 293 cells. They
conclude that the Asp 298 residual is directly
involved in MC4R adenylyl cyclase signalling. How-
ever, no differences in ligand binding were observed
between both variants. Wikberg et al. (2000) con-
cluded that leptin signalling can be alternatively
mediated via an MC4R-independent path because
MC4R-deficient mice do still respond to leptin. Barb
et al. (2004) also conclude from their results in pigs
that the action of leptin on feed intake may be medi-
ated by a regulatory mechanism circumventing the
melanocortin 4 receptor. Their data do not support
the idea that the Asp298Asn mutation in MC4R leads
to a loss of function. The existence of alternative sig-
nalling cascades may thus be an explanation for the
contradictory effects obtained for the functional poly-
morphism of MC4R in different pig lines. Park et al.
(2002) detected two suggestive quantitative trait loci
(QTL) for fatness traits flanking the position of MC4R
on chromosome 1. This supports the hypothesis that
gene interactions may modify the observed direc-
tion of the linkage between MC4R variants and
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R. Jokubka et al.
performance traits. The use of breeding values on the
basis of estimations of genetic parameters for the
entire Lithuanian White population instead of single
measurements in our study reduces the bias in the
recorded performance traits. However, as the poten-
tial effect of a candidate gene variant on the perform-
ance trait is included in the estimated breeding value
for the animals already, a post hoc comparison of can-
didate variants underestimates the candidate gene
effect (Israel & Weller 1998). Therefore, the signifi-
cant results obtained for the population of Lithuanian
White endorse the integration of the MC4R genotype
into selection programmes for increasing growth rate
and reducing fatness of this breed.
Acknowledgements
For the enabling to access the database, S. Rimkevi-
cius, head of the State Pig Breeding Station, is
acknowledged. Financial support was provided by the
Animal Husbandry and Veterinary Division at The
Ministry of Agriculture of the Republic of Lithuania.
The authors wish to thank Dr V. Saferis (Institute for
Biomedical Research, Kaunas University of Medicine)
for excellent statistical advice and assistance.
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