Relationship between Myosin Isoforms and Meat Quality Traits in Pig Semitendinosus Neuromuscular Compartments

: The aim was to determine the relationship between muscle structure and meat quality traits in neuromuscular compartments (NMCs: R1, R2, R3, R4) of pig semitendinosus muscle. Barrows from the INTA-MGC genetic line (Argentina) were slaughtered at 100 kg body weight. In each NMC the following parameters were determined: the fibre types I, IIA, IIX and IIB by immunohistochemistry, the fibre cross sectional area (FCSA), the pH of meat after 24 h post-mortem (pH 24 ), instrumental meat tenderness (WB) and colour (L*, a*, b*). There were significant differences in the following: L* (R1 = R4<R2 = R3), a* (R1>R4>R2 = R3), b* (R1 = R4<R2 = R3), WB (R2>R1 = R3 = R4), pH 24 (R1 = R4>R2 = R3). The relative percentages of FCSA were as follows: I (R4>R1>R3>R2), IIA (R1>R4>R3>R2), IIX (R1 = R2 = R3 = R4) and IIB (R2>R3>R1>R4). The correlation values were statistically significant between IIB and WB (R1 and R4, r s = 0.66), (R2 and R3 r s = 0.74), IIB and L* (R1 and R4 r s = 0.84), IIX and L* without discriminating NMCs. Our data suggest that the NMC where the sampling takes place is important for determining meat quality traits because of the heterogeneity of the whole muscle.


INTRODUCTION
Meat quality can be evaluated by several traits, such as pH 24 , the instrumental meat tenderness by Warner-Bratzler shear force (WB) and colour (L*, a* and b*). Intrinsic muscle characteristics, such as fibre types, the fibre cross sectional areas (FCSA), perimortem metabolism, and environmental components including feeding and rearing system have an impact on the change from muscle to meat by modifying the values of the meat quality traits (Karlsson et al., 1999;Park et al., 2007;Nam et al., 2009). Furthermore, it is important to note that mammalian skeletal muscles are not homogeneous. Mammalian skeletal muscles are composed of neuromuscular compartments (NMCs) defined by the distribution of primary nerve branches that may alter the muscle fibre characteristics and the metabolic profile (Roy et al., 1984;Bruce et al., 1993;Kernell, 1998;Graziotti et al., 2004;Graziotti et al., 2009). The myosin heavy chain (MyHC) isoforms, I, IIa, IIx and IIb (with decreasing oxidative capacity in that order), are expressed in pig skeletal muscles (Quiroz-Rothe and Rivero, 2004) and vary significantly among different NMCs (Graziotti et al., 2008). The relationship between muscle fibre types and meat quality in pigs is inconsistent and not completely understood (Gentry et al., 2004) probably for not to keep in mind the muscular partitioning in heterogeneous NMCs. In this study the aim was to investigate in each NMC the relationship between the fibre characteristics and meat quality traits.

Gross anatomy
To identify the NMCs, in a previous assay (Graziotti et al., 2009) the left semitendinosus (ST) muscle was removed from five carcasses preserving the sciatic nerve branches. The muscles were cleaned of fat and connective tissues, immersed in a 10% formalin solution for 30 days, rinsed with tap water for 3 days and incubated with a 25% nitric acid solution for 10 days while checking the muscles every 48 h during the entire procedure. Macroscopic dissections were carried out following the primary branches of the sciatic nerve ( Figure 1).

Immunohistochemistry
This research was carried out on 12 right ST muscles of barrows (n = 12) from the INTA-MGC genetic line (Argentina) slaughtered at 100 kg body weight. Muscle samples for immunohistochemistry were taken from the core of each NMC (R1, R2, R3 and R4) 3 h postmortem, and the samples were frozen by immersion in liquid nitrogen and kept at -80°C until analysis. To identify the fibre types, 10 μm thick serial sections were obtained using a cryostat at -27°C, and the sections were then mounted on glass slides and reacted by adenosine triphosphatase myofibrillar (mATPase) after acid preincubation (pH 4.6) (Brooke and Kaiser, 1970) modified by Nwoye et al. (1982). Other serial sections were incubated with a panel of monoclonal antibodies (MAbs) that were specific for MyHC isoforms (Table 1). The immunohistochemical procedure with the avidin-biotin peroxidase complex (ABC) was used for the localisation of primary antibody binding as described Graziotti et al. (2004). In brief, sections were preincubated in a blocking solution of stock goat serum. After the primary antibody was diluted in Phosphate Buffered Saline, the antibody was applied to the sections for 40 min in a humid chamber at 37°C. The sections were then washed and incubated with a secondary antibody. After incubation with the secondary antibody, the sections were washed and incubated with the ABC reagent. Diaminobenzidine tetrahydrochloride was used as a chromogen to localise the peroxidase. The proportion of hybrid fibres was irrelevant (<3.5%), but the immunohistochemically delineated fibre types were characterised as the pure fibre types (I, IIA, IIX and IIB) according to Abreu et al. (2006).
For quantitative studies the FCSA were determined by captured images in a TIFF format employing the Motic Image Plus 2.0 software. The FCSA of each individual fibre type was determined in the slides treated with mATPase with an image (10×magnification) by drawing a mask along the cell borders (100 fibres at least). Opensource software (Scion Image beta 3b) was utilised for determining the FCSA.

Statistical analysis
Statistical analysis was carried out by Friedman's test, being the NMCs the treatments and the animals the blocks and p<0.05 was considered statiscally significant. The Spearman's test was used for the correlation analysis.
The correlation between the fibre type IIB and the WB value was statistically significant with regard to the R1 and R4 NMCs (r s = 0.66) as well as the R2 and R3 NMCs (r s = 0.74). The correlation between the fibre type IIB and L* value was statistically significant with regard to R1 and R4 (r s = 0.84). The correlation between the fibre type IIX and L* value was statistically significant without discriminating NMCs.

DISCUSSION
The ST muscle, a strongly partitioned muscle, was used for this study because it has the phenotypic expression of the I, IIa, IIb and IIx MyHC isoforms (Lefaucheur, 2006).
In accordance with Park et al. (2007) our results indicate that the MyHC isoforms, expressed in each of the studied NMCs, are relevant attributes in meat quality development because they influence perimortem metabolism. The meat quality trait values in each of the oxidative NMCs (R1 and R4) and glycolytic NMCs (R2 and R3) correspond to the fibre muscle characteristics in glycolytic or oxidative muscles that were determined in prior investigations (Gentry et al., 2004;Hu et al., 2008;Gil et al., 2008).
The higher a* values and the lower L* and b* values in the R1 and R4 NMCs are in agreement with a relationship among a* values, red fibre percentages (I-IIa MyHC isoforms) and oxyhaemoglobin values. (Park et al., 2007;Kwasiborski et al., 2008;Gil et al., 2008;Bérard et al., 2008;Nam et al., 2009).
Such as red fibres contain higher myoglobin values, the pH values decline slowly. This may explain the greater pH 24 values in the R1 and R4 NMCs when compared to the R2 and R3 NMCs (Kwasiborski et al., 2008). Similarly, the smaller values found in the R2 and R3 NMCs may be explained by the greater rate and longer period of postmortem glycolysis in these two NMCs (Bee et al., 2006).
Although the relation between the WB values and muscle fibre characteristics is unclear (Sazili et al., 2005), a positive correlation among the WB values, FCSA and percentage of type IIB fibres was suggested (Hu et al., 2008;Nam et al., 2009). In accordance, the results in this study indicated that the FCSA of the type IIB (Table 3) and the WB value (Table 2) were significantly higher in the R2 NMC when compared to the other NMCs.
According to Gentry et al. (2004) muscular structure in animal husbandry research is not completely understood. Some authors (Cerisuelo et al., 2007;Hu et al., 2008) use numerous sampling places to correct variations due to the muscle heterogeneity (Algañaraz, 2007). The heterogeneity among muscle NMCs requires accurate anatomical references for muscle sampling because the conclusions based on a single muscle sampling location are not representative (Janz et al., 2006;Lefaucheur, 2006). A standard sampling following neuromuscular partitioning may be more appropiate because previous muscular architecture studies (Graziotti et al., 2009) indicate that NMCs are composed of muscular fibres with homogeneous characteristics.
We conclude that sampling according to the distribution of primary nerve branches while maintaining the threedimensional orientation and the relation with anatomical references is an appropriate tool for examining the entire muscle.

ACKNOWLEDGMENTS
This research was supplied by Grant V-803 UBACYT, Buenos Aires University, Argentina.