The Effects of Stress Related Genes on Carcass Traits and Meat Quality in Pigs

The current study was conducted to investigate the relationship between stress related gene and meat quality in pigs. A total number of 212 three-way cross bred (Landrace-Yorkshire×Duroc) and 38 Duroc were sampled from the Korean pig industry to determine genotype frequency of porcine stress syndrome (PSS) and heat shock protein 70 kDa (HSP70) genes and their relationship with carcass traits and longissimus meat quality. Screen of HSP70 was performed by the single strand conformation polymorphism (SSCP) technique. Based on the analysis of restriction fragment length polymorphism (RFLP) in ryanodine receptor 1 (RYR1) gene, genetic disorder of PSS was related to a mutation at 18,168 (C to T) of exon 17. There was no significant difference in ultimate meat pH and backfat thickness between HSP70 K1-AA type and -BB type in pure Duroc breed. In Landrace-Yorkshire×Duroc (L-YxD) cross bred pig, our results indicated that HSP70 derivate type in Duroc had a limited effect on backfat thickness, but L-YxD type had a noticeable linkage with HSP70 K1-AA and K3-AB. This tendency was also observed in hot carcass weight where HSP70 K1-AA and K3-AB resulted in heavier weight with 86.3 kg compared to HSP70 K1-AB and K3-BB of 74.3 kg. Results imply that stress related HSP70 genotype has a potential association with backfat thickness and carcass weight. (Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 2 :


INTRODUCTION
Extensive research has been performed to reduce the most notorious quality defect of pale, soft and exudative (PSE) pork over last decades (Forrest et al., 1997;Rosenvold and Andersen, 2003;Kim et al., 2005).It has been identified that PSE meat is related to a fast glycolysis during the onset of rigor under which condition protein denaturation occurs (Cassens et al., 1975) which consequently reduce water holding capacity (WHC) and juiciness (Cheah et al., 1998;Gispert et al., 2000).
On the other hand, genetic component has been shown as another significant factor determining ultimate meat quality through its interactions with extrinsic components (Riëtte et al., 1993).The genetic influence on pork quality exists between breeds and within a breed.The variation is caused by a large set of genes known as polygenic effects, and in principle most traits of interest for meat quality have a multifactorial background (McPhee and Trout, 1995;Gispert et al., 2000;Andersson, 2001).Fujii et al. (1991) reported that a point mutation in pig RYR1 gene is associated with PSS, and RYR1 gene is a major gene known to have a direct linkage with pork quality (Rosenvold and Andersen, 2003).The causative mutation for the halothane (HAL) gene is in the gene encoding for a pig RYR1 isoform.The HAL or stress gene effect has been well documented since the 1991 discovery of a DNA test for direct identification of genotypes (Fujii et al., 1991).
In general, homozygous and heterozygous pigs for the HAL gene have higher carcass yield and lean percentage (Simpson and Webb, 1989;McPhee and Trout, 1995;Larzul et al., 1997).However, the positive effect of the HAL gene on performance is counterbalanced by its negative effect on WHC and colour.As indicated by name, PSS carrying pigs are highly susceptible to stress.These animals are genetically more susceptible to stress even during normal shipping and handling.The hypermetabolism is increased levels of lactic acid, CO 2 , heat and oxygen consumption (Forrest et al., 1997).Even with a particular care, a limited stress prior to slaughter is sufficient to trigger a higher rate of post mortem glycolysis in both homozygous and heterozygous pigs, being most severe in the homozygous pigs (Mitchel and Heffron, 1982;Rosenvold and Andersen, 2003).
Heat was the first factor known to induce the formation of these stress proteins, which subsequently became known as heat shock proteins (HSP).These proteins are found in several protein families, with 70-kDa (HSP70) and 90-kDa (HSP90) families, being the most abundant (Riëtte et al., 1993).Cells or multi-cell organisms respond to heat or other stresses by inducing or increasing the synthesis of HSP (Lindquist and Craig, 1988;Welch, 1992).In the unstressed cell, HSP70 is diffusely located in both the cytoplasm and the nucleus, but in the heat shocked cell there is a concentration of HSP70 in nucleoli, as well as an increase of the protein in both cytoplasm and nucleus (Welch and Feramisco, 1984).The transient expression of HSP70 in the piglet is likely attributed to periods of increased muscular activity, or the increased metabolic demands (McComb and Spurlock, 1997).Kilgore et al. (1994) suggested that circumstances that fit either an increase or decrease in muscle mass may invoke expression of HSP70.Stress response in organisms has been a subject of extensive scientific investigation.Riëtte et al. (1993) investigated the pork quality and expression of stress protein HSP70 in swine.There was no difference in the HSP70 express patterns of tissues from HAL-positive and HAL-negative pigs.The pig that showed the strongest expression of HSP did not appear to be related to the meat quality characteristics from stressed pigs.DNA polymorphisms of PSS and HSP70 gene might be due to individual difference of pork quality from preslaughter treatment (Christian and Lundstrom, 1992;Houde et al., 1993;Riëtte et al., 1993;Rosenvold and Andersen, 2003).Therefore, the purpose of this study was to determine the effects of RYR1 and HSP70 genes on carcass characteristics in pigs.

Animals and DNA sampling
Number of animals and genetic characteristics were described in Tables 1 and 2. M. longissimus samples were taken from two hundred and twelve pigs from commercial abattoir (Han Naeng Co. LTD., Ochang, Chungbuk) in Korea, while 38 blood samples were collected from the National Livestock Research Institute (NLRI).Genomic DNA was isolated from both whole blood and muscle samples, and purified according to the method of Sambrook et al. (1989).The isolated DNA was diluted to a final concentration of 50 ng/ml.

Meat quality measurements
The carcass weight (kg) and the tenth backfat thickness (cm) were measured on the hanging carcasses.Longissimus thoracis ultimate pH was evaluated at 24 h post mortem.The day after slaughter, longissimus muscles (from the last thoracic vertebrae to the last lumbar vertebrae) were taken from left side of each carcass, and prepared for the measurements of objective meat colour, Warner-Bratzler (WB) shear force and cooking loss.WB-shear force was measured on steaks (2.54 cm thick) cooked in a pre-heated water bath for 60 min until the core temperature had reached 70°C and then cooled in running water (ca.18°C) for 30 to reach a core temperature below 30°C.Eight cores of 1.27 cm diameter were made for each sample, and peak force was determined using a V-shaped shear blade with a cross-head speed of 400 mm/min (Wheeler et al., 2001).Cooking loss was determined by calculating the weight difference in steaks before and after cooking, expressed as percentage of initial weight.Objective meat colour was determined by a Minolta Chromameter (CR300, Minolta, Japan) on freshly cut surface after a 30 min blooming at 4°C.

Statistical analysis
The effects of PSS and HSP70 genotypes on objective meat qualities were evaluated by analysis of variance using GLM procedure of SAS/STAT.The model included HSP70 K1 genotype effect for pure Duroc Breed and PSS-HSP70 K1-HSP70 K3 for three-way cross bred.Differences in least squares means were analysed by Fisher's least significant difference test (SAS, 1990) with a comparison error rate of 0.05.

Genotyping
As shown in Figure 1, PSS genotyping with HhaI RFLP showed that HhaI (C/C) PCR products exhibited two fragments of 145 bp and 76 bp (normal pig).HhaI (T/T) PCR products exhibited one fragment of 221 bp (mutant pig).HhaI (C/T) PCR product exhibited three fragments of 221 bp, 145 bp and 76 bp (carrier pig).Three other genotype alleles were observed after HhaI digestion of the 221 bp PCR-amplified fragment.The normal C/C allele of PSS digested and produced fragments of 145 and 76 bp.The mutant T/T allele of PSS did not cut but produced a 221 bp fragment.The PCR-SSCP polymorphisms of pig HSP70 segments for K1 (223 bp, 290-512) and K3 (595 bp, 830-1,424) were detected and the mutant band consisted of AA, AB and BB (Figure 2).The three genotypes (AA, AB and BB) alleles were obtained at 10°C, 30% MDE gel using PCR amplification of HSP70 K1 290-512 (2a) and HSP70 K3 830-1,424 (2b) fragments.

Duroc pure breed meat quality
No significant differences were observed in carcass and objective meat quality characteristics among the three PSSfree HSP70 K1 types (AA, AB and BB) in Duroc pure breed (Table 1).There was, however, a consistent tendency of the genotypes to have linear effects on meat quality traits.For example, the least square means showed that ultimate pH, WHC and meat colour values increased with the expression of type B (i.e. from AA to AB to BB).In contrast, WB-shear force and cooking loss displayed a reverse trend (i.e.decreasing from AA to AB to BB) (Table 1).The ultimate pH value recorded in the current study for type BB (5.92) was relatively higher than those reported for normal pig longissimus muscle in other studies (De Smet et al., 1996;Depreux et al., 2002).It is generally known that ultimate pH in pig muscle is a reflection of many confounded factors including feeding regime, transit stress and slaughtering process and that has a significant effect on meat quality (Hwang et al., 2004), but the higher pH for the currently used animal group was not clearly identified.However, results of PSS and HSP70 genotypes showed no significant difference for ultimate pH (Table 2).Similarly, Klont et al. (1993) did not find any significant difference for ultimate pH between carriers and normal genotypes.The range in WB-shear force values in our investigations (2.50-2.98 0.5 in 2 /kg) were lower than those observed by Moelich et al. (2003) in PSS genotype group (3.0-3.2 kg/1.27cm).

Backfat thickness and carcass weight
Our results indicate that HSP70 genotype from Duroc had a limited effect on meat quality characteristics but L-Y×D crossbred had a noticeable linkage with HSP70 K1-AA and K3-AB.This effect was observed in the hot carcass weight where significantly (p<0.05)higher weight was recorded in HSP70 K1-AA and K3-AB (86.3 kg) compared to HSP70 K1-AB and K3-BB (74.3 kg) (Table 2).Consequently, backfat thickness was also higher in HSP70 K1-AA and K3-AB (2.40 cm) at p<0.05 than other groups, although significant terms were not found, indicating a relationship between backfat thickness and carcass weight.Therefore, the data imply that heavier carcass weight had higher backfat thickness although in some cases this is variable.This trend also suggests that stress-related HSP70 genotype could be associated with backfat thickness and carcass weight.In spite of the limited number of pigs for some genetic groups, the trend corroborates observations by Gispert et al. (2000) that PSS-carrier yielded heavier carcass weight and thicker backfat.Other studies have, however, showed little but non-significant effects of stressrelated HSP70 genotype on backfat thickness (McPhee and Trout, 1995;Nanni Costa et al., 2002).

Loin weight
Left loin and right tender loin weights were significantly (p<0.05)influenced by stress-related genotype (Table 2).The left loin weight for the PSS-Carrier pigs with genotype HSP70 K1-AB and K3-AB (3.32 kg) was significantly higher than that for the PSS-Normal genotype HSP70 K1-BB and K3-AB (2.91 kg).The PSS-Normal genotype HSP70 K1-BB and K3-AB had the highest yield of tenderloin (442.9 g) while the lowest tenderloin yield was produced by the PSS-Normal genotype HSP70 K1-AB and K3-AB (434.5 g).Leach et al. (1996) reported significantly (p<0.01)lower Least Mean Squares for wholesale loin cut weights for heterozygous (normal carrier) pig than for the homozygous normal.

Meat colour
The CIE lab values (L*, a* and b*) for the genotypes shown in Table 1 revealed no significant difference (p>0.05) for the three genotypes.Various studies have shown that higher L* value are as a result of PSE meat (Lawrie, 1991).Meat from HSP70 KI-BB genotype had a higher mean reflectance value (L* = 52.39)indicating pale meat compared with both AA (L* = 51.7) and AB (L* = 51.8)genotypes.Studies done by Bartone et al. (1988) revealed that colour changes in meat are associated with abnormal pH values and water holding.This is consistent with these study where ultimate pH and water holding capacity was also similar in trend with L* values.However, contrast patterns were observed in PSS carrier genotype (Table 2) which had a lower L* value (46.3) than PSS normal genotype (48.4).In addition, the b* value was significantly (p<0.05)lower in PSS carrier HSP70 K1-AB and K3-AB (1.92) than in PSS Normal (ranging from 2.79 to 3.62).However, there is need to increase the sample size of the PSS carrier to determine accurately the CIE values for comparison purposes.The results obtained in this study, therefore, indicate that there are significant effects on meat quality due to meat colour as described by Brewer and McKeith (1999).The colour of meat is an excellent indicator of pork quality and pale 'very light pink' is regarded as inferior quality indicating the presence of PSE condition.Taken together, we have investigated the effect of stress-related gene of PSS and HSP70 gene on carcass traits and meat quality.These genes were detected in the genotype through the PCR-RFLP and -SSCP from each amplified DNA fragments.In particular, the genotypes of PSS and HSP70 gene have a potential association with backfat thickness and carcass weight.These results can apply to the pig breeding by utilizing polymorphisms detection.

Figure 1 .
Figure 1.The PCR-RFLP analysis of RYR1 gene.Lane 1, standard markers pUC18/HaeIII; Lanes 2 and 3, DNA from a C/C pig; Lanes 4 and 5, DNA from a T/T pig; Lanes 6 and 7, DNA from a C/T pig; Lane 8, PCR products that amplified from 18,475 to 18,695.

Table 1 .
Effects of HSP70 K1 genotype on carcass and meat quality traits of Duroc pure breed RFLP analysis 10 µl of the 221 bp PSS fragment was digested with 5 units of HhaΙ at 37°C for 2 h.The digested DNA fragment was then separated by electrophoresis on 12% polyacrylamide gel in 1×TBE.The gel was stained with ethidium bromide (2 µg/ml) and visualized under UV light.The HSP70 genotypes of PCR products were separately confirmed by PCR-SSCP.For SSCP analysis, 8 µl of PCR products were mixed with 8 µl of loading buffer Science Rockland, Inc., ME, USA) gel (1.0 mm) with 0.7×TBE.The gel was stained with ethidium bromide and visualized under UV light.

Table 2 .
Effects of PSS and HSP70 genotype on carcass and meat quality traits of Landrace-Yorkshire×Duroc pigs 1 Ultimate (at 24 h post mortem) pH values in the loin.a, b, c LSM±SE with different superscripts in the same row are significantly different (p<0.05).