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Meat Quality Traits Associated With the Malignant Hyperthermia (MH) Genotype in
Sweedish Landrace Pigs in Croatia
Zoran Tadić, Vesna Benković, Mirko Lojkić1, Ivan Bašić
Department of Animal Physiology, Faculty of Science, Rooseveltov trg 6, HR-10000 Zagreb,
Croatia
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Croatian Veterinary Institute, Savska 143, HR-10000 Zagreb, Croatia
Summary
We investigated the effect of the malignant hyperthermia gene status on the some meat
quality characteristics in 339 pigs of Sweedish Landrace breed fattened to 105 kg. Pigs of nn
genotype had higher fat thickness, and lower loin eye depth than the NN and Nn animals.
Drip loss was the lowest in the NN and the highest in the nn animals. Measurements of pH
showed significantly lower pH in the nn pigs, as compared to the NN and Nn pigs. Since pH
after one hour post slaughtering was less that 6.00 in the nn pigs, the quality of their meat falls
within the category of the PSE meat. The nn animals also had paler meat than their NN and
Nn counterparts. Since the Nn pigs are not leaner than the NN pigs, as revealed by their fat
thickness and loin eye depth, the heterozygotic effect on the improvement of meat quality is
doubtful. Moreover, drip loss of these animals lies somewhere between the NN and nn
animals which shows the negative effects of the mutated RYR1 gene on the meat quality. We,
therefore, suggest, that the mutated RYR1 gene should be used cautionously in pig breeding,
as its effects may not always be favourable.
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Souhrn:
Prozkoumali jsme vliv genů maligní hyperthermie na některé charakteristiky kvality
masa u 339 prasat rodu švedský Landras odchovaných do váhy 105 kg. Prasata genotypu nn
měla větší tloušťku tukové vrstvy a menší tloušťku slabinového svalu, než NN a Nn prasata.
Ztráta tekutiny z masa (angl. drip loss) byla nejmenší u NN a největší u nn zvířat. Měření pH
ukázala značně nižší pH masa u nn prasat, než u NN a Nn prasat. Jelikož pH masa byl 1
hodinu po porážce nižší než 6.00, patří kvalita tohoto masa do kategorie PSE. Recesní nn
zvířata měla také světlejší maso, než zvířata ganotypů NN a Nn. Jelikož se podle těchto testů
Nn prasata neukázala štíhlejší než NN prasata, je kontrolované vnášení mutovaného RYR1
genu v chovu pochybné. Kromě toho, se změřené hodnoty ztráty tekutiny z masa Nn zvířat
nacházejí někde mezi hodnotami NN a Nn zvířat, což ukazuje na negativní účinek
mutovaného RYR1 genu na kvalitu masa.
Doporučujeme proto, aby se vnášení mutovaného RYR1 genu při chovu prasat
provádělo velice pozorně, protože jeho účinky nemusí být vždy pozitívní.
Key words: pigs, malignant hyperthermia, RYR1 gene, meat quality
Introduction
Malignant hyperthermia (MH) or porcine stress syndrome (PSS) is a genetic disease of
swine which is usually expressed under stress conditions (e.g. bad farming practice,
inappropriate transport of the animals) or during inhalation of anaesthetics. The disease is
characterized by muscular cramps, reddening of the skin and high body temperature. The gene
responsible for the disease is called RYR1 and is situated on the short arm of the sixth
chromosome (Harbitz et al., 1990). It encodes a channel protein which is crucial for the
regulation of the flow of the Ca2+ ions during muscular contraction cycle (MacLennan and
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Phillips, 1992). Eleven years ago, a new test based on the amplification of the affected portion
of the gene by polymerase chain reaction has been devised (Fuji et al., 1991; Otsu et al.,
1992). This test proved to be much reliable than the previously used halothane inhalation test,
as it was able to detect all three RYR1 genotypes. Moreover, this test allowed quick testing of
a large number of swine in a short time (O'Brien et al., 1993).
The malignant hyperthermia has very important consequences for the pig farming and
meat industry. Deregulation of Ca2+ traffic during contraction leads to the osmotic imbalance
and causes redistribution of water between cytoplasm and the extracellular fluid. The result is
pale, soft, exudative (PSE) meat, which is not suitable for the market. Complete elimination
of the mutated RYR1 gene proved to be undesirable, as animals that are heterozygous (Nn) are
generally leaner, faster growing and have an increased feed conversion efficiency, as
compared to dominant homozygous pigs (Murray et al., 1989; Leach et al., 1996). However,
Nn pigs are also four times more likely to produce PSE pork than NN pigs, as they are more
susceptible to stress (Barton-Gade et al., 1988).
High production efficiencies with concomitant increase in leanness and growth rate
stimulated some producers to incorporate mutated RYR1 gene in breeding herds, despite the
known effects in the pork quality. The rationale of this approach is to enable producers to
target carcass specifications of high value according to price.
The extent of spread of the mutated RYR1 gene in the population of breeding swine in
Croatia is unknown. There has never been systematic halothane testing and, consequently,
possible correlation of the meat quality traits with the RYR1 genotype remains unknown. A
few years ago, we did a pilot study of genetic testing of swine with the aim of ascertaining its
feasibility in Croatia (Lacković et al., 1997). In this work, we wished to assess possible
changes in meat quality traits, which may be associated with the malignant hyperthermia
genotypes in Sweedish landrace pigs raised on a farm in Croatia during the fattening process.
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Materials and methods
We tested 339 animals of Sweedish Landrace breed from three big farms in Croatia.
Among them were 104 castrated males and 235 gilts. All the animals were fattened to
approximately 105 kg..The animals were transported from the farms to the abattoir by road.
They were weighted immediately prior to slaughtering which was done according to
commercial procedures. Pigs were stunned, exsanguinated and dehaired. Following
evisceration, carcasses were weighted, split down the midline and put in the chiller at 4oC.
The carcasses remained in the chiller for approximately 24 hours.
Measurements of pH were done approximately 45 minutes (pH1) and 24 hours (pH24)
after slaughtering at the level of the last rib using portable pH meter (Corning pH/ion meter)
fitted with spear – type ion electrode. Each day, pH meter was calibrated using pH 6.00 and
pH 8.00 buffers. Drip loss was measured in musculus longissimus dorsi samples. The samples
(approx. 100 grams) were removed from the carcass 24 hours post - slaughter. The muscles
were removed of all fat and each 85 g sample was suspended in the air – filled, labelled
plastic bag kept at 3oC for 48 hours (Murray et al., 1989). Excess moisture was lightly
removed from the muscle surface and samples were reweighed. Drip loss was calculated and
expressed as percentage of total weight. Water – holding capacity (WHC) was determined by
the filter paper pressing method (Charpentier et al., 1971). If the value was greater than 2.4,
the WHC was considered poor. Meat colour was assessed on longissimus dorsi muscle 24
hours post – slaughter. We used the meat colour scale described by Nakai et al. (1975). The
scale is divided in 6 levels: 1 – very pale meat, 2 – pale meat, 3 – light pink meat, 4 – dark
pink meat, 5 - dark meat, 6 – very dark meat. Since meat colour assessment is a procedure,
which heavily depends on the experience of the person and subjective reckoning, it was done
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independently by three persons. The final result was obtained by calculating the mean of three
observations. Fat thickness was measured at the level of 3rd – 4th posterior rib.
Molecular analyses were done in the laboratory on the blood samples taken at the
farm. We collected about 0.5 ml of blood by ear venipuncture. The blood was collected into
1.5 ml sterile Eppendorf tubes containing 200 l of EDTA (1.5 mg/ml). For the determination
of the RYR1 genotype we used the method described by Otsu et al. (1992), with the slight
modification in PCR cycling parameters done by O'Brien et al. (1993). Briefly, the
erythrocytes were lysed in 0.5 ml of TE buffer, pH 8.0. The mixture was centrifuged for two
minutes at 13500 rpm/room temperature in the “Eppendorf 5415” tabletop centrifuge. The
resultant pellet was washed three times with 0.5 ml of TE buffer and digested with proteinase
K at 56oC/90 minutes. The proteinase K was inactivated at 96oC/10 minutes and 5 l of this
lysate was used for PCR amplification. The amplification was done in the total volume of 25
l. The mixture contained DNA, PCR buffer, MgCl2, dNTPs and “AmpliTaq DNA
Polymerase”. The primers used for the amlification were 5’-TCCAGTTTGCCACAGGTC
CTACCA-3’ (forward) and 5’-TTCACCGGAGTGGAGTCTCT-3’ (reverse). The
amplification cycle was: Denaturation at 94oC/13 seconds; primer annealing and extension at
67oC/80 seconds. Total number of cycles was 30 after which the mixture was cooled at 4oC.
After amplification, 1 l of the mixture was digested with Bsi HKI restriction enzyme in total
volume of 10 l and the resulting fragments were resolved on a 3% agarose gel. The
frequency of the mutated allele was calculated using the following equation:
frequency = [(No. of nn homozygotes X 2) + No. of heterozygotes)] / No. of tested
animals X 2
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Before and after the slaughtering process, we checked all the animals for the presence
of specific tattoo marks on the ears to identify them and exclude possible misidentification.
Statistical analyses were done using multifactorial ANOVA with sex and genotype
being crossed factors (Iles, 1993). We used the following general linear model (GLM):
Yij =  + Gi + Sj + GSij + ij
Where Yij is dependent variable,  is the overall mean, Gi is the genotype effect, Bj is
the sex effect, GBij is the interaction between the genotype and the sex, ij is the residual. The
probability of rejection of H0 was less than 5% (p < 0,05). Analyses were done with the SPSS
10.0 Statistical Software (SPSS Inc., Chicago, USA).
Results and discussion
Of 339 animals, 22 (6.48%) were heterozygous (Nn), 10 were recessive homozygotes
(2.95%) and the remainder were dominant homozygotes. The frequency of the mutated RYR1
allele was calculated to be 0.062.
We did not observe any significant differences for sex within genotype on the meat
quality traits (results not shown). All the other results are sumarized in Table 1.
The nn pigs had significantly thinner fat, as compared to the NN and Nn pigs. These
results are comparable the results of some authors (Pommier et al., 1992; Dovč et al., 1996;
Garcia-Marcias et al., 1996; de Smet et al., 1998) which showed that the carcasses of the nn
pigs have the lowest fat thickness, with Nn intermediate and NN the highest. Some authors,
however, showed that nn pigs have significantly thicker fat that the NN pigs (Fisher and
Mellet, 1997). We also showed that gilts had significantly thinner fat that castrates. Since we
also find that the interaction of the sex and genotype is not significant, it may be tempting to
examine the sex and genotype effects separately. Various authors used different pig breeds in
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their studies and it is also possible that the RYR1 gene may manifest itself differently in
different breeds, causing various fat thickness.
The loin eye was significantly deeper in the NN and Nn than in the nn pigs. However,
there is no significant difference in the loin eye depth between the sexes, although castrates
have a slightly deeper loin than the gilts. It seems that the inclusion of the mutated RYR1 gene
does not have effects on the leanness of the Nn pigs. If this was the case, loin eye depth of
these animals should be significantly higher than that of NN animals. Fat thickness should
also be lower than that in the NN animals. On the other hand, it may be that this is just
reflection of farming practice connected with the influence of feeding quality.
We observed significant differences in drip loss between NN, Nn and nn pigs, the nn
pigs having the highest and the NN pigs the lowest drip loss. There were no significant
differences in drip loss between the sexes. Since malignant hyperthermia symptoms are the
result of the uncoordinated calcium flow in the muscle, flooding of myocytes with calcium
induces osmotic imbalance and uncontrolled water flow in muscle. Such animals have
elevated drip loss, the Nn animals having the intermediate drip loss between the nn and NN
animals. The same applies to the water – holding capacity of the muscle which showed
increasing difference from NN to nn animals, the nn animals having the poorest water –
holding capacity. The Nn animals have the water – holding capacity which lies between the
NN and nn pigs. Since malignant hyperthermia cause great water movements in the muscle,
the poor water – holding capacity of the nn pigs is not surprising.
Meat colour also shows differences between the genotypes, but not between the sexes.
The NN pigs have the darkest meat while the nn pigs have the palest. The changes in the
RYR1 genotype may profoundly influence fresh pork quality and this is comparable to the
results of other authors who suggest that meat colour changes are due to colour destruction by
myoglobin autoxydation (Tam et al., 1998). This effect is the most profound in the nn pigs.
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Measurements of pH showed that the NN pigs have significantly higher pH1 than the
nn pigs, the Nn animals being somewhere in between. There were no significant differences
between the sexes. However, as the pork quality may also be influenced by other factors, such
as the pre – slaughter handling and stunning method (Channon et al., 2000), it may be
possible that early post – mortem pH changes are also influenced by this factors, at least to
some extent. We also noted the same difference in pH24 between the three genotypes. These
differences are not so profound as the differences in pH one hour after slaughtering, but still
are statistically significant. It is interesting that the pH of the meat of the Nn pigs dropped
slightly below 5.5 which is the isoelectric point of the main muscle proteins (Lawrie, 1984).
This should worsen the characteristics of the PSE meat such as the drip loss. The reason why
the pH drop was more pronounced in Nn than in nn pigs remains unclear and warrants further
investigation.
Some breeders encourage the incorporation of the mutated RYR1 gene into the
breeding stock, as Nn pigs are shown to be generally leaner, faster growing and have better
food conversion efficiency, as compared to the NN pigs. However, they are also more likely
to produce PSE meat. Our results suggest that this may not be the best breeding strategy, as
many meat quality traits are inferior in Nn animals, as compared to the NN animals. Since
meat quality traits may differ in various pig breeds and they may express various mutated
RYR1 frequencies, wider investigation is underway to determine the extent of the spread of
the mutated RYR1 gene in the population of the breeding swine in Croatia and its effects on
fattening and meat quality traits.
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These experiments were done as part of the project “Determination of the
Malignant Hyperthermia Gene Status in Swine in Croatia” (project No. 119001)
funded by the Ministry of Science and Technology of the Republic of Croatia.
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Corresponding author:
Zoran Tadić
Department of Animal Physiology
Faculty of Science
University of Zagreb
Rooseveltov trg 6
HR-10000 Zagreb
Croatia
tel. +385 1 482 6266
fax. +385 1 482 6260
E-mail: ztadic@public.srce.hr