The effect of calpastatin polymorphism and its interaction with RYR1 genotypes on carcass and meat quality of crossbred pigs

The aim of the study was to establish the relationship between a calpastatin gene (CAST) polymorphism, the ryanodine receptor gene (RYR1) polymorphism and carcass/meat quality traits in crossbred pigs. No significant differences in the analyzed pigs were found between genotypes CC and CT at the locus RYR1 and CD and DD at the locus CAST/MspI in terms of carcass and meat quality. However, a significant association of the CAST/ApaLI polymorphism with carcass quality and meat marbling were observed. The carcasses of AB pigs had significantly higher carcass percentage of lean meat, thinner backfat and thicker muscle, as well as lower meat marbling, as compared with the BB pigs. Furthermore, interactions CAST/MspI × RYR1 and CAST/ApaLI × RYR1 were found significant in relation to all the studied carcass traits. The results presented here imply that the CAST gene recognized with ApaLI may be considered as important in terms of the way it affects porcine carcass quality traits. Moreover, the research has revealed a relationship between CAST and RYR1 genotypes as regards formation of carcass traits in pigs. Follow-up studies, however, should be carried out on larger populations representing all possible CAST genotypes.


Introduction
Intensive efforts on improvement of swine percentage of lean meat with use of Pietrain pigs resulted in a number of issues, especially those linked to a high frequency of the stress syndrome gene (RYR1 T ) observed in this breed, which is associated with occurrence of pale, soft, and exudative (PSE) meat (Fiedler et al. 2001). Moreover, research shows, despite belonging to very different breeds, pigs of the same genotype for the RYR1 gene not only do exhibit a considerable variability in carcass lean content, but also provide meat of varying quality. This may be an effect of other genes that possibly affect both carcass traits and meat quality, modifying the effect of the RYR1 (Koćwin-Podsiadła and . Calpastatin (CAST) is the endogenous inhibitor involved in regulation of calpain activity in muscle cells. Activity of calpains and calpastatin relies on appropriate concentration of calcium ions in the cell (Murachi 1989). Moreover, calpastatin activity is strongly correlated with muscle growth rate, proteolytic processes, and immediate post mortem changes in the muscle (Goll et al. 1998), which affects many quality traits of the meat Melody et al. 2004).
The calpastatin gene has been mapped near the centromere of SSC 2 in the region q2.1-q2.4. The calpastatin molecule consists of domain L, coded by exons 2 to 8, and four repetitive domains, each of which is coded by exons 9-14 (Stearns et al. 2005). Polymorphisms in the calpastatin gene (CAST), identified in the intron with 3 restriction enzymes, HinfI, MspI, and RsaI, were first reported by Ernst et al. (1998). Ciobanu et al. (2004), on the other hand, identified a CAST gene polymorphism at domains L, 1, and 4, recognized using ApaLI, Hpy188I, and PvuII enzymes. Chromosome 2 may be associated with the QTL related to tenderness and shear force because the QTL affiliated with the traits were relatively close in position and shared similar effects Ciobanu et al. 2004). The QTL associated with fat percent was within the same marker inter-val as the QTL related to shear force, suggesting pleiotropic or linked QTL (Malek et al. 2001).  and Krzęcio et al. (2004) demonstrated that a number of meat quality traits significantly depended on the animal's genotype at the locus CAST. The authors state that such correlation can be used in swine selection for better carcass and meat quality. In our studies, on the same cross pigs, in which to identify calpastatin gene (CAST) polymorphism were used restriction enzymes HinfI and Hpy188I, was found significant association between calpastatin gene (CAST/HinfI) and meat quality traits (Rybarczyk et al. 2010).
The aim of the study was to find a possible relationship between variants of the calpastatin gene (CAST/MspI and CAST/ApaLI), the ryanodine receptor gene (RYR1) and carcass/meat quality traits in crossbred pigs sired by Pietrain boars.

Material
The experiment was carried out on 125 pigs (76 gilts and 49 barrows) from a pig farm located in Mecklemburg-Vorpommern (Germany). The study comprised offspring from crossing German Landrace × German Large White and also Leicoma × German Large White sows with Pietrain boars, kept under similar environmental conditions and fed with a balanced feed-mix ad libitum. All subjects destined for the experiment were conveyed in one group to the "Agryf" Meat Plant in Szczecin (Poland) in the evening after 4 hours transportation from a distance of 250 km, and slaughtered on the next day in the morning.

Slaughter value
After animal CO 2 stunning during the slaughter, blood was collected for identification of CAST and RYR1 genotypes. Subsequently, carcass percentage of lean meat was measured, hot carcass weight, as well as the thickness of the longissimus dorsi (LD) muscle and back fat between the 3rd and 4th rib, 7 cm from the line of carcass partition into the left-hand side of the carcass half, by means of an optic-needle CGM apparatus (Sydel, France). Mean percentage of lean meat amounted to 55.39 ± 0.40 and hot carcass weight to 87.75 ± 0.55 kg (mean value and standard error).

Meat quality and basic meat chemical composition
Two hours after slaughter during carcass cooling, electric conductivity (EC 2 ) was measured in the longissimus dorsi muscle, between the 4th and 5th lumbar vertebrae of the right-hand side of the carcass half using an LF-Star MATTHÄUS conductometer. After 24 hours of carcass cooling, meat samples from the longissimus dorsi muscle were collected from the 1 st to-4 th lumbar vertebrae section (longgisimus lumborum) of the right-hand side of the carcass half. 24 hours from the slaughter, meat pH 24 value (Elmetron CP-311 pH-meter) and the volume of drip loss from the muscle tissue were determined according to Honikel (1987). Within 48 hours after slaughter, minced meat samples were measured for pH in water solution, and meat colour traits, i.e. L* (lightness), a* (redness) and b* (yellowness), were established by means of a HunterLab Mini Scan XE Plus 45/0 with light illuminant D65 and observer 10 º (CIE 1976). Meat water-holding capacity (WHC) was determined according to Grau and Hamm (1952) as modified by Pohja and Niinivaara (1957), as well as thermal drip from a difference of meat sample weight before and after heating in a water bath at 85 º C for 10 minutes. Water-soluble protein content was determined by Kotik method (1974). Marbling (the degree of intramuscular fatness) was determined by a trained 5 person team of panellists, using a 1-5 point scale (1 point -slight muscle fatness; 5 points -strong muscle fatness). The basic meat chemical composition, i.e. total protein, fat, ash and dry matter (AOAC 2003), was estimated.

Statistical analysis
A statistical analysis was performed to compare carcass and meat quality traits and basic chemical composition of meat between pigs of different CAST and RYR1 genotypes, using the least squares method of the GLM procedure (Statistica 8.0 PL) according to the following linear model: where: Y ijkl -trait measured, μ -the overall mean, a i -the effect of sex (i = 1, 2), b j -the effect of RYR1 genotype (j = CT, CC), c k -the effect of CAST/MspI genotype (k = CD, DD) or CAST/ApaLI genotype (k = AB, BB); bc jk -interaction (RYR1 × CAST/MspI or CAST/ ApaLI genotype), β -linear regression coefficient for hot carcass weight; x ijkl -hot carcass weight of ijkl-th individual included as covariable; x -mean for hot carcass weight; e ijkl -the random error.
A detailed comparison of mean least squares (LSQ) for the analysed CAST and RYR1 genotypes was done using a Tukey's test.

Results
The frequencies CAST/MspI, CAST/ApaLI, and RYR1 of alleles and genotypes in Pietrain-sired pigs are presented in Table 1. The significances of association between the genotypes of calpastatin (CAST/MspI and CAST/ApaLI), RYR1 and carcass and meat quality traits of the pigs are presented in Table 2. We did not find any significant differences between the genotypes CC and CT of the RYR1 in terms of carcass percentage of lean meat, backfat and LD muscle thickness, as well as in meat quality and basic chemical composition determined in the longissimus lumborum muscle.
Significant differences were found in carcass slaughter performance and meat marbling between pigs of genotypes AB and BB at the locus CAST/ ApaLI (Table 3). The AB pigs had significantly higher percentage of lean meat, thinner backfat, thicker LD muscle, and lower marbling of the meat as compared with the BB genotype (p ≤ 0.05). No significant association, however, was found between the CAST/ApaLI genotype and meat quality traits of the pigs. Moreover, significant CAST/ ApaLI × RYR1 interactions were found in terms of carcass quality. Significantly higher percentage of lean meat and thinner backfat (p ≤ 0.01), as well as thicker LD muscle (p ≤ 0.05), were found in the AB/CT, AB/CC, and BB/CT genotypes in relation to BB/CC pigs (Table 4).
No significant effect of the CAST/MspI polymorphism on carcass and meat quality or meat basic chemical composition was observed. Significant CAST/MspI × RYR1 interactions, however, were found as regards carcass quality of the pigs. The DD/CC and CD/CT pigs had significantly higher percentage of lean meat (p ≤ 0.01) and thinner backfat (p ≤ 0.05) as compared with the CD/ CC genotype. Moreover, DD/CC pigs had a significantly thicker LD in relation to CD/CC (p ≤ 0.05).

Discussion
The presented study on Pietrain-sired pigs of unknown family structure did not reveal significant differences in carcass or meat quality between CC and CT genotype at the locus RYR1. Results by other authors who studied carcass and meat quality in the same genotypes of pigs with Pietrain genes are not unambiguous. Busk et al. (2000) stated that the carcasses of CT pigs contained more lean and had worse meat quality as compared with the CC pigs. Kusec et al. (2005) did not observe significant differences in butchery value of carcasses between the CC and CT pigs; the authors found, however, that meat quality of heterozygous pigs (CT) was worse. Also Krzęcio et al. (2005) and Kuhn et al. (2005) report poorer quality of meat obtained from CT pigs. On the other hand,  did not find significant differences in meat quality between the discussed RYR1 genotypes. The analysis of CAST/MspI genotypes frequency in the studied crossbred pigs sired by Pietrain boars revealed two genotypes, CD and DD, which was also reported by Kurył et al. (2003) in pigs obtained from the program TORHYB [Pietrain × (Polish Landrace × Polish Large White)] as well as in Polish Landrace pigs. In both the cited report and studies by Ernst et al. (1998), Pietrain pigs were monomorphic at the locus CAST/MspI and were of DD genotype. All three possible genotypes were observed in Yorkshire and Large White pigs (Ernst et al., 1998), as well as in Stamboek (Dutch Large White × Dutch Landrace) and Złotnicka Spotted .
We did not observe significant differences in carcass quality between pigs of CD and DD genotypes at the locus CAST/MspI. Conversely,  demonstrated a relationship between the CAST/MspI polymorphism and some carcass quality traits in RYR1 T -free Stamboek hogs. DD pigs had thinner backfat at some measurement points and a larger rib eye area in relation to CC pigs. Also Koćwin-Podsiadła et al. (2004) observed that crossbred, RYR1 T -free fatteners revealed significant effect of association between the CAST/MspI genotype and five out of 19 analyzed carcass traits. The authors report that activity of a given molecular type of calpastatin depends on the muscle, since BB-genotype pigs at CAST/MspI had a larger ham weight, whereas larger loins were cut from AA pigs.
The analyzed pigs did not show a significant association between the CAST/MspI genotype and meat quality as well as basic chemical composition of meat in the longissimus lumborum muscle. Also  and Kurył et al. (2004), who studied crossbreds sired by Duroc × Pietrain boars, failed to demonstrate a relationship between the CAST/MspI genotype and meat quality traits, except for loin efficiency during smoking. Krzęcio et al. (2005), on the other hand, observed significant effects of the CAST/MspI polymorphism on the level of lactic acid in the longissimus lumborum muscle tissue 45 minutes post mortem, index of energy metabolism intensity R 1 , water holding capacity (WHC), drip loss from muscle tissue at 48 and 96 hours post mortem, as well as meat protein and water content. Moreover, Koćwin-Podsiadła and  demonstrate that polymorphism at the locus CAST/MspI relatively strongly correlates with the incidence of increased drip loss from meat in the group of pigs with the gene RYR1 T .
We have demonstrated here a significant association between the CAST/ApaLI genotype and carcass quality traits and meat marbling. Bonforroni's correction revealed a significant linkage between the CAST/ApaLI and percentage of lean meat. It is known that the proteolytic calpain-calpastatin system has its role in the processes of muscle growth and development. Results of experiments show that the calpain system is important for the proper development of the skeletal muscles. An increased rate of skeletal muscles development may be a result of a reduced rate of protein degradation in the muscle, which is associated with a reduced activity of the calpain system resulting mainly from a considerable increase in calpastatin activity (Goll et al. 1998). The presented results may indicate that calpastatin as calpains inhibitor may behave differently depending on the genetic variant. Moreover, analysis found significant evidence of QTL on SSC 2 associated with backfat thickness, longissimus muscle area and fat percent (Malek et al. 2001;Stearns at al. 2005).
The analysis of the pigs revealed significant effect of interactions, CAST/ApaLI × RYR1 and CAST/MspI × RYR1, in relation to carcass quality traits only, i.e. percentage of lean meat, backfat and LD muscle thickness. Bonforroni's correction revealed significant interactions between CAST/MspI × RYR1 in relation to percentage of lean meat and muscle thickness, as well as between CAST/ApaLI × RYR1 in relation to percentage of lean meat and backfat thickness. On the other hand, Krzęcio et al. (2005), who studied a group of crossbred pigs, observed a significant effect of interaction between the RYR1 and CAST/MspI genotype for muscle tissue acidity 24 hours post mortem and drip loss from the LL tissue 48 hours post mortem. In the studies on crossbred pigs sired by Duroc × Pietrain boars, an interaction between the CAST/MspI and RYR1 genotypes was significant only for loin efficiency during smoking  and drip loss measured at 48 hours post mortem ). The authors Krzęcio et al. 2005) conclude that the frequency of meat with high drip loss in pigs free of the stress sensibility gene (genotype CC at the locus RYR1), as well as that of normal meat in carriers of the gene (genotype CT at the locus RYR1) may be a result of a joint modifying effect of the CAST gene on the post mortem changes in the muscle tissue.

Conclusions
No significant differences in the analyzed pigs sired by Pietrain boars were found between genotypes CC and CT at the RYR1 locus and CD and DD at the CAST/MspI locus in terms of carcass and meat quality. However, a significant influence was found of the CAST/ApaLI polymorphism on carcass quality and meat marbling. The carcasses of AB pigs had significantly higher percentage of lean meat, thinner backfat, and thicker LD muscle, as well as lower meat marbling, as compared with the BB pigs. Furthermore, interactions CAST/MspI × RYR1 and CAST/ApaLI × RYR1 were found significant in relation to all the studied carcass traits, i.e. percentage of lean meat, backfat thickness, and LD thickness.
The results presented here imply that the CAST gene recognized with ApaLI may be considered as important in terms of the way it affects porcine carcass quality traits. Moreover, research has revealed an association between CAST and RYR1 genotypes as regards formation of carcass traits in pigs. Follow-up studies, however, should be carried out on larger populations representing all possible CAST genotypes.