Relationships between the polymorphism of myosin heavy chains and selected meat quality traits of pigs with different susceptibility to stress

The aim of the investigations was to analyse the share of myosin heavy chains (MHC) isoforms (type I, IIa, IIb, and IIx) in the longissimus thoracis et lumborum muscle derived from pigs of different RYR1 genotypes (TT – homozygous negative, CT – heterozygous, CC – homozygous positive). The composition of the MHC isoforms in the muscle tissue of the examined animals was referred to selected meat quality traits. It was revealed that the animals with the CT and TT genotypes were characterized by a significantly (P≤0.05) lower share of the type I and higher share of the type IIb MHC isoform in comparison to homozygotes CC. Inferior tenderness and water holding capacity of meat obtained from pigs susceptible to stress (TT) at 144 h after slaughter could have been associated, among others things, with the increased share of MHC isoform type IIb. The composition of MHC isoforms might be a useful indicator in breeding work in the selection of animals carrying the gene of susceptibility to stress.


Introduction
Quality of pork depends on many factors, among which a special role is played by genetic factors (SELLIER and MONIN 1994, VON LENGERKEN et al. 1994, DE VRIES et al. 2000).
A major gene determining meat quality and susceptibility of pigs to stress is the ryanodine receptor gene (RYR1), previously known as the halothane gene (HAL) (FUJII et al. 1991, SELLIER and MONIN 1994, LUNDSTRÖM et al. 1995, DOVC et al. 1996, KOĆWIN-PODSIADŁA et al. 2000, KRZĘCIO et al. 2004, KUSEC et al. 2005, WOJTYSIAK and MIGDAŁ 2007).A mutated allele of RYR T is responsible for the occurrence of the PSE defect of meat, especially in pigs with high meat contents in the carcass.An additional adverse effect of these anomalies is the occurrence of malignant hyperthermia (the MH syndrome), resulting in the denaturation of contractile protein (myosin) and sarcoplasmic protein (myoglobin), and thus in the deterioration of many meat quality attributes (BOLES et al. 1991, LUNDSTRÖM et al. 1995, MARTENS 1998).
The adverse effect of the RYR1 gene on meat quality, as well as its processability and eating quality, was confirmed in numerous studies (BOLES et al. 1991, DE SMET et al. 1998, ESSÉN-GUSTAVSSON et al. 1992, LUNDSTRÖM et al. 1995, GÖDEKE et al. 1998, THALLER et al. 2000, KRZĘCIO et al. 2004).Significant dependencies were found between traits of muscle fibres, susceptibility to stress and meat quality (ESSÉN-GUSTAVSSON et al. 1992, FIEDLER et al. 1993, 2001, PEDERSEN et al. 2001, DEPREUX et al. 2002, EGGERT et al. 2002, RYU and KIM 2005).The discovery of immunohistochemical methods made it possible to distinguish types of muscle fibres based on specific isoforms of myosin heavy chains (MHC) (SCHIAFFINO and REGGIANI 1996, GREASER et al. 2001, PONSUKSILI et al. 2008) and relate them to muscle quality.
The aim of the study was to analyze the proportions of isoforms of myosin heavy chains (MHC) in m. longissimus thoracis et lumborum collected from pigs with different RYR1 genotypes.The composition of MHC isoforms in the muscle tissue of tested animals was referred to selected quality attributes of their meat.

Material and methods
The experimental material was m. longissimus thoracis et lumborum, which was collected from a total of 66 porcine half-carcasses, including 36 crossbred fatteners (Landrace × Yorkshire) × Duroc [(L×Y)×Dur] and 30 fatteners of Line 890 (Line 990 × Pietrain) with varied susceptibility to stress.The parental generation of (L×Y)×Dur crosses was imported from Denmark.Porkers of Line 890 were obtained from the Experimental Farm, Pawłowice, and were maintained at the Experimental Animal Nutrition Farm, Gorzyń belonged to Poznań University of Life Sciences.Feeding of animals from which muscles were collected, as well as their management conditions, were controlled.Genotypes of analyzed animals in terms of locus RYR1 were identified using PCR-RFLP (FUJII et al. 1991).A total of 30 animals were diagnosed with genotypes CC, 18 -with genotype CT, and 12 -with genotype TT.Only (L×Y)×Dur crosses were free from the gene of susceptibility to stress.
Pigs were slaughtered under standard abattoir conditions using electrical stunning.Samples (2-5 g) for electrophoretic analyses were collected 45 min after slaughter from the longissimus muscle and stored at −80 °C until analyzed.The remaining part of the analyzed muscle after cooling was divided into two halves, of which the thoracic section (thoracis) was used in analyses after 48 h, while the lumbar section (lumborum) 144 h after slaughter.Prepared portions (of 400-600 g) after vacuum packaging were transported to the laboratory under cold-storage conditions in order to assess tenderness and water holding capacity of meat from analyzed animals.
Electrophoretic analyses concerned the determination of isoforms of myosin heavy chains (MHC).Their separation in the fraction of washed myofibrils collected from the muscle tissue 45 min after slaughter was run in 8 % PAGE-SDS (MOZDZIAK et al. 1998) with an SE 260 apparatus by Hoefer Scientific Instruments.Washed myofibrils were collected as a result of double washing of the muscle tissue with a phosphate buffer solution, the rigor buffer (75 mM KCL, 10 mM KH2PO4, 2 mM MgCl2, 2 mM EGTA with pH 7.0, 0.1 M PMSF) (FRITZ et al. 1989).Each sample transferred onto gel contained 0.5 μg protein.
Electrophoresis was run at a constant voltage of 70 V at 4 °C for 24 h.After the separation was completed gels were stained in a solution containing 0.05 % Coomasie blue R-250, 45 % methanol and 9.2 % acetic acid and next destaining in a mixture of 10 % methanol and 7.5 % acetic acid.Quantitative analysis of separated bands of MHC isoforms was conducted with an Image Master VDS scanning densitometer by Pharmacia, using Image Master1D Elite version 4.00 software.Computations were based on the assumption that the area of a single protein band accounts for a percentage ratio in relation to the area of all separated proteins in a given sample on gel, which constitutes 100 %.
Instrumental assessment of meat tenderness was conducted on the basis of shear force required to cut 10×10 mm samples in an Instron 1140 universal testing machine with a Warner-Bratzler device (WBSF), following thermal processing.Meat slices with a thickness of 25-30 mm were heated in a Rational Combi convection oven in hot air at 160 °C for approx.15 min (GRZEŚ et al. 2005).
Sensory examination of meat tenderness on heated meat samples was conducted using the linear scaling method (ADAMIK 1997, BARYŁKO-PIKIELNA 1990).Desirability scores were analyzed in a 0-10 point scale (»0« corresponded to very tough meat, »10« to very tender meat).
Water holding capacity was determined based on centrifugal and thermal drip from the muscle tissue (HONIKEL 1987) as a result of centrifugation (at 25 000× g for 20 min at 20 °C) or heating, respectively.The volume of drip, expressed in per cent, was calculated from the difference of meat weights before and after centrifugation or heating.
Results were subjected to a one-way analysis of variance (ANOVA) in an nonorthogonal system due to the unequal number of animals in the treatments using Statistica 8.0 PL software (STANISZ 2000).The significance (P≤0.05) of differences between the animal groups segregated according to the RYR1 genotypes for individual meat quality traits was determined using the Fisher test.

Results
Results of the analysis of variance presented in the table showed a statistically significant (P≤0.05)effect of the gene of susceptibility to stress (RYR1) on the composition of muscle fibres corresponding to specific isoforms of myosin heavy chains (MHC), as well as tenderness and water holding capacity of meat in case of analyzed pigs.
Out of the analyzed four types of MHC isoforms (I, IIa, IIb, and IIx) the content of only two of them, i.e.IIb and I, was significantly (P≤0.05)varied between analyzed RYR1 genotypes (Table ).At the same time it was observed that the proportion of MHC isoform type IIb (corresponding to fast glycolytic fibres) was significantly (P≤0.05)higher in the groups of CT (68.87 %) and TT fatteners (68.87 %) in comparison to CC animals (62.76 %).In turn, shares of MHC isoform type I (corresponding to slow oxidative fibers) was significantly (P≤0.05)lower in the group of RYR1 T carriers (3.24 % for CT, 3.70 % for TT) in comparison to animals which were not carriers of this gene (9.33 %).
Analysis of meat tenderness both in case of instrumental and sensory examination showed significant (P≤0.05)dependencies only at 144 h after slaughter (Table ).At that time point a significantly (P≤0.05)lower shear force value, i.e. superior tenderness, was recorded for stress resistant animals (CC) (33.79 N/cm 2 ) in comparison to both CT (47.33 N/cm 2 ) and TT groups (47.61 N/cm 2 ).In turn, in sensory examination of tenderness significant (P≤0.05)differences were found between TT (5.63 points) vs. CT (6.98 points) and CC genotypes (7.31 points).Analysis of water holding capacity based on the volume of centrifugal and thermal drip showed statistically significant (P≤0.05)dependencies both at 48 h and 144 h after slaughter (Table ).In that case volumes of centrifugal drip were significantly (P≤0.05)varied between TT (after 48 h -22.51 %, after 144 h -17.57%) and CC homozygotes (after 48 h -20.22 %, after 144 h -13.66 %).In turn, volumes of thermal drip were significantly (P≤0.05)varied between CC (after 48 h -24.75 %, after 144 h -23.99 %) and CT (after 48 h -29.76 %, after 144 h -29.56 %) and TT groups (after 48 h -31.03 %, after 144 h -30.55 %).The lowest volumes of centrifugal and thermal drip were recorded for meat sampled from stress resistant animals (Table ).

Discussion
In porcine skeletal muscles four types of muscle fibres (I, IIA, IIB and IIX) are distinguished, in which specific isoforms of myosin heavy chains (MHC) are found (SCHIAFFINO and REGGIANI 1996, DEPREUX et al. 2002, EGGERT et al. 2002, CHANG et al. 2003, MELODY et al. 2004, DA COSTA and CHANG 2005, RYU and KIM 2005, CHOI et al. 2007, WOJTYSIAK and MIGDAŁ 2007).Type I fibres are known as slow oxidative fibres, type IIB -as fasttwitch glycolytic fibres, while types IIA and IIX -as intermediate, oxidative glycolytic fibres.Electrophoretic separation of MHC isoforms in the analyzed porcine muscle (m.longissimus thoracis et lumborum) showed the presence of four types of MHC isoforms.At the same time, we need to stress here the unseparated bands of MHC isoforms types IIa and IIx (Figure).The above phenomenon was also observed in a study by MELODY et al. (2004).It results from earlier studies that individual types of fibres mature in a specific order (I ↔ IIA ↔ IIX ↔ IIB) (PETTE and STARON 1997) and later maturating fibers as well as fibres with a larger cross-section area are related, among things, with deteriorating meat quality (LEFAUCHEUR and GERRARD 2000).In this study a higher proportion of MHC isoforms type IIb was recorded in the group of pigs -RYR1 T carriers (TT and CT).At the same time meat sampled from that group of animals was characterized by inferior tenderness and water holding capacity in comparison to animals which did not carry the gene of susceptibility to stress.This probably resulted from an increased amount of fast glycolytic fibres, as it was previously indicated also by other researchers (ESSÉN-GUSTAVSSON et al. 1992, DEPREUX et al. 2002, EGGERT et al. 2002, FIEDLER et al. 2001, WOJTYSIAK and MIGDAŁ 2007).At the same time they observed that the effect of fibre types on meat quality in pigs was more evident in the group of RYR1 T carriers than in the group of animals which did not carry that gene.Thus it became possible to use the composition of MHC isoforms in the selection of animals exhibiting susceptibility to stress (MARTENS 1998, DEPREUX et al. 2002, EGGERT et al. 2002, DA COSTA and CHANG 2005, LEFAUCHEUR 2006, CHOI et al. 2007).
Conducted studies showed a statistically significant (P≤0.05)effect of the RYR1 gene on the proportion of isoforms of myosin heavy chains (MHC) type IIb (corresponding to fast glycolytic fibres) and type I (corresponding to slow oxidative fibres) in the muscle tissue of tested fatteners.
Carriers of the RYR1 T gene (TT and CT) were characterized by a significantly (P≤0.05)lower share of MHC isoforms type I and a higher proportion of MHC isoform type IIb in comparison to animals resistant to stress (CC).
Inferior tenderness (higher values of shear force in instrumental analysis and lower scores in sensory examination), as well as water holding capacity (bigger volume of centrifugal and thermal drip) of meat collected from pigs susceptible to stress (TT) at 144 h after slaughter could have been related, among other things, with increased contents of MHC isoforms type IIb.
The composition of MHC isoforms might be a useful indicator in breeding work in the selection of animals carrying the gene of susceptibility to stress.

Figure
Figure Electrophoretic separation of MHC isoforms in the longissimus thoracis et lumborum muscle obtained from pigs of different RYR1 genotypes (8 % PAGE-SDS) Elekrophoretische Trennung der MHC Isoformen im Schweinemuskel (M.longissimus thoracis et lumborum) mit verschiedenen RYR1 Genotypen (8 % PAGE-SDS) Mean values from the same row with various letters differ statistically significantly at P≤0.05.