Association of RYR 1 and MYOG Genotype with Carcass and Meat Quality Traits in Grower-finisher Pigs

The study involved 110 hybrid grower-finisher pigs from Polish Large White × Polish Landrace sows sired by Pietrain breed boars and their crossbreds with the 990 line and Duroc. The aim of the study was to determine an association of the polymorphism of RYR1, myogenin (MYOG) genes and carcass meatiness level with carcass slaughter value and meat quality traits in hybrid pigs. Better meat quality was found in pigs of CC genotype at locus RYR1 than in those of CT genotype, with a similar carcass slaughter value and meat chemical composition. No significant differences were found in meat traits (carcass slaughter value) and quality between AA and AB genotypes at locus MYOG. Similarly, no significant differences were found between carcass meatiness ranges adopted in meat quality and its chemical composition, except for water holding capacity, which was higher in pigs with ≤ 54% meatiness than in those with > 54%. Moreover, interaction was found between carcass meatiness range and RYR1 genotype in relation to backfat thickness and intramuscular fat content, as well as a connection between carcass meatiness range and MYOG genotype in relation to water-soluble protein content. The results indicate the need to continue selection work towards the elimination of allele T RYR1 gene’s in pedigree herds to improve meat quality in fatteners. Grower-finisher pigs, myogenin, RYR1, meatiness, meat quality Studies indicate that pigs belonging to different breeds but characterised by the same genotype at the RYR1 locus show significant differences in carcass meatiness and meat quality. The occurrence of PSE meat among pigs of CC genotype or the occurrence of normal meat in animals of TT genotype may be a consequence of the effect of other genes on carcass and meat quality traits modifying the effect of RYR1 genotype (Koćwin-Podsiadła and Kurył 2003). It has been found that some other genes or their families could be singled out as so-called candidate genes with a potential effect on carcass meatiness based on their participation in the processes of skeletal muscle development in the foetal period. These genes include the MyoD family genes, i.e. MYOD1 (MYF3), MYF5, MYOG (MYF4) and MYF6 (MRF4) (Te Pas and Visscher 1994). It has been shown that significance of the relationship between the value of some carcass traits and MYOG genotype was breeddependent (Kurył et al. 2002; Cieślak et al. 2002). Studies of the aforementioned authors and those of Krzęcio et al. (2007a) showed that MYOG genotype was associated with the formation of selected carcass quality traits, whereas those of Kapelański et al. (2005) and Krzęcio et al. (2007b) also pointed to a link with pork quality. On the other hand, Urbański et al. (2007) did not find significant relationships between MYOG genotype and the structure of porcine longissimus dorsi muscle. Moreover, Horák et al. (2004) showed an association between MYOG gene polymorphism and reproductive traits in Přeštice Black-Pied breed pigs. This study was aimed at determining an association of the polymorphism of RYR1, ACTA VET. BRNO 2010, 79: 243-248; doi:10.2754/avb201079020243 Address for correspondence: Artur Rybarczyk, PhD Department of Livestock Product Evaluation Faculty of Biotechnology and Animal Science Westpomeranian University of Technology Str. Doktora Judyma 24, 71-466 Szczecin, Poland Phone +48 091 449 6708 Fax +48 091 454 1642 E-mail: Artur.Rybarczyk@zut.edu.pl http://www.vfu.cz/acta-vet/actavet.htm myogenin (MYOG) and carcass meatiness level genes with carcass slaughter value and meat quality traits in hybrid grower-finisher pigs. Materials and Methods The study was carried out on 110 hybrid grower-finisher pigs, including 57 barrows and 53 gilts, from Polish Large White × Polish Landrace sows sired by Pietrain boars and their crossbreds with 990 line and Duroc. The animals were individually kept and fed at a pig farm at the Experimental Station of Animal Production (National Research Institute of Animal Production) in Kołbacz, Poland. The energy value and basic chemical composition of feed mixture used in the study was consistent with the Pig Nutrition Standards of 1993. After reaching the body weight of 100 ± 2 kg, the pigs were slaughtered and blood samples were collected for DNA analysis to identify all genotypes at loci RYR1 and MYOG. During slaughter operation, meat acidity (pH1) was measured 45 min after bleeding (pH-meter CP-311, Elmetron) in the longissimus dorsi (LD) muscle between the 4th and the 5th lumbar vertebrae of the right half-carcass; warm carcass weight was also determined. The mean carcass weight was 81.02 ± 1.33 kg. Thereafter, following a 24-hour chilling, the carcasses were dissected according to the methods used at Swine Testing Stations (Różycki 1996) and the meat pH24 was determined. The dissection results were then used for the calculation of the carcass lean content. Hybrid porker carcasses were divided into two groups according to their meatiness: I ≤ 54% (n = 61) and II > 54% (n = 49). During cutting, meat samples were collected from the LD muscle from the lumbar vertebrae 1-4 section of the right half-carcass. Approximately 48 h after slaughter, pH measurement was done in water solution on mixed and diluted muscle tissue, meat colour lightness (L*) was determined by means of HunterLab Mini Scan XE 45/0 apparatus (CIE 1976), water holding capacity was determined by the Grau-Hamma method as modified by Pohja and Niinivaara (1957), thermal drip according to Walczak (1959), water-soluble protein content by Kotik’s method (1974), as well as the assay of meat basic chemical composition, i.e. total protein, fat, ash, and dry matter (AOAC 1990). Examination of the tenderness of the longissimus dorsi muscle (last four thoracic vertebrae) was performed by means of an Instron 1140 Universal Testing Machine (Instron, High Wycombe, UK) using the Warner-Bratzler test. The DNA analysis, by PCR/RFLP technique, was performed at the Institute of Genetics of the Mendel University of Agriculture and Forestry in Brno, Czech Republic. The RYR1 genotypes were identified by means of HinPI endonuclease (Fujii et al. 1991), whereas the polymorphism of MYOG gene by means of the restriction enzyme MspI in the 3’ region (Soumillion et al. 1997). The statistical analysis was performed to compare carcass and meat quality traits and meat basic chemical composition between pigs of different RYR1 and MYOG genotypes and meatiness group, using the least squares method of the GLM procedure (Statistica 7.1 PL) according to the following linear model: Yijklm = μ + ai + bj + ck + kl + bcjk + bkjl + ckkl + eijklm where: Yijklm – trait measured, μ – overall mean, ai – effect of sex (i = 1, 2), bj – effect of RYR1 genotype (j = CT, CC), ck – effect of MYOG genotype (k = AA, AB); kl – effect of meatiness group (l = I, II); bcjk – effect of interaction between RYR1 and MYOG genotype; bkjl – effect of interaction between RYR1 genotype and meatiness group; ckkl – effect of interaction between MYOG genotype and meatiness group; eijklm – random error. Detailed comparison of mean least squares (LSQ) for the analysed RYR1 and MYOG genotypes and meatiness group was done using Tukey test. Results and Discussion Two alleles, C and T, were identified in the analysed pigs that determined the occurrence of two genotypes, CT and CC, in locus RYR1 (Table 1). The frequency of the C allele was 0.65, whereas that of the T allele was 0.35. Based on the analysis of genotypes in locus 244 Table 1. Frequency of RYR1 and MYOG genotypes and allele in pigs

myogenin (MYOG) and carcass meatiness level genes with carcass slaughter value and meat quality traits in hybrid grower-finisher pigs.

Materials and Methods
The study was carried out on 110 hybrid grower-finisher pigs, including 57 barrows and 53 gilts, from Polish Large White × Polish Landrace sows sired by Pietrain boars and their crossbreds with 990 line and Duroc.The animals were individually kept and fed at a pig farm at the Experimental Station of Animal Production (National Research Institute of Animal Production) in Kołbacz, Poland.The energy value and basic chemical composition of feed mixture used in the study was consistent with the Pig Nutrition Standards of 1993.
After reaching the body weight of 100 ± 2 kg, the pigs were slaughtered and blood samples were collected for DNA analysis to identify all genotypes at loci RYR1 and MYOG.During slaughter operation, meat acidity (pH 1 ) was measured 45 min after bleeding (pH-meter CP-311, Elmetron) in the longissimus dorsi (LD) muscle between the 4 th and the 5 th lumbar vertebrae of the right half-carcass; warm carcass weight was also determined.The mean carcass weight was 81.02 ± 1.33 kg.Thereafter, following a 24-hour chilling, the carcasses were dissected according to the methods used at Swine Testing Stations (Różycki 1996) and the meat pH 24 was determined.The dissection results were then used for the calculation of the carcass lean content.Hybrid porker carcasses were divided into two groups according to their meatiness: I ≤ 54% (n = 61) and II > 54% (n = 49).During cutting, meat samples were collected from the LD muscle from the lumbar vertebrae 1-4 section of the right half-carcass.Approximately 48 h after slaughter, pH measurement was done in water solution on mixed and diluted muscle tissue, meat colour lightness (L*) was determined by means of HunterLab Mini Scan XE 45/0 apparatus (CIE 1976), water holding capacity was determined by the Grau-Hamma method as modified by Pohja and Niinivaara (1957), thermal drip according to Walczak (1959), water-soluble protein content by Kotik's method (1974), as well as the assay of meat basic chemical composition, i.e. total protein, fat, ash, and dry matter (AOAC 1990).Examination of the tenderness of the longissimus dorsi muscle (last four thoracic vertebrae) was performed by means of an Instron 1140 Universal Testing Machine (Instron, High Wycombe, UK) using the Warner-Bratzler test.The DNA analysis, by PCR/RFLP technique, was performed at the Institute of Genetics of the Mendel University of Agriculture and Forestry in Brno, Czech Republic.The RYR1 genotypes were identified by means of HinPI endonuclease (Fujii et al. 1991), whereas the polymorphism of MYOG gene by means of the restriction enzyme MspI in the 3' region (Soumillion et al. 1997).
The statistical analysis was performed to compare carcass and meat quality traits and meat basic chemical composition between pigs of different RYR1 and MYOG genotypes and meatiness group, using the least squares method of the GLM procedure (Statistica 7.1 PL) according to the following linear model: where: Y ijklm -trait measured, μ -overall mean, a i -effect of sex (i = 1, 2), b j -effect of RYR1 genotype (j = CT, CC), c k -effect of MYOG genotype (k = AA, AB); k l -effect of meatiness group (l = I, II); bc jk -effect of interaction between RYR1 and MYOG genotype; bk jl -effect of interaction between RYR1 genotype and meatiness group; ck kl -effect of interaction between MYOG genotype and meatiness group; e ijklm -random error.
Detailed comparison of mean least squares (LSQ) for the analysed RYR1 and MYOG genotypes and meatiness group was done using Tukey test.

Results and Discussion
Two alleles, C and T, were identified in the analysed pigs that determined the occurrence of two genotypes, CT and CC, in locus RYR1 (Table 1).The frequency of the C allele was 0.65, whereas that of the T allele was 0.35.Based on the analysis of genotypes in locus RYR1, a higher percentage of heterozygous individuals was found (70.9%), and lower of the CC homozygotes (29.1%).The analysis of alleles in locus MYOG showed a higher frequency of the allele A (0.91) and lower of the allele B (0.09), which determined the occurrence of two genotypes, AA and AB.The percentage of the AA porkers was 82.7%, whereas of the AB it was 17.3%.The carcass meatiness ranges adopted in the present study (≤ 54% and > 54%) significantly differentiated the analysed porker carcasses with respect to meatiness, backfat thickness and the loin muscle eye area (P ≤ 0.01), which is presented in Tables 2 and 4. The analysis of meat quality and its basic chemical composition showed significant differences only in meat water-holding capacity (WHC), which was favourably higher in the carcasses with meatiness below 54% (P ≤ 0.05).Krzęcio et al. (2005) showed a negative effect of higher carcass meatiness on meat quality.On the other hand, Rybarczyk et al. (2006) did not find any differences in meat quality and its chemical composition according to carcass meatiness level in their studies on pigs from sows of white breeds and Pietrain boars.
The results presented in Table 2 show that no significant differences were found in the analysed hybrid pigs in carcass slaughter value traits and meat basic chemical composition according to RYR1 genotype (CT and CC), which is also confirmed by studies of other authors (Fisher et al. 2000;Kusec et al. 2005).However, no positive effect of the  Smet et al. 1996;Janik et al. 2006), was confirmed.When analysing the quality of meat in pigs differentiated by the RYR1 genotype, it was shown that the meat of the CC genotype pigs was characterised by higher pH, darker colour (L*) (P ≤ 0.05), lower thermal drip, higher waterholding capacity (WHC) and higher water-soluble protein (P ≤ 0.01) compared to those of CT genotype (Tables 3 and 5), which corroborates the findings pointing to the negative effect of the T allele on meat quality (Oliver et al. 1993;Biedermann et al. 2000) but is not confirmed in the study of Koćwin-Podsiadła et al. (2003), which shows a similar meat quality of the pigs of CC and CT genotypes.Furthermore, no interaction was found between the meatiness range and RYR1 genotype in the hybrid pigs in relation to meat quality traits, which was also observed by Krzęcio et al. (2005) in a study on pigs with 25% of Pietrain breed and by Rybarczyk et al. (2006) in a study on pigs with 50% of Pietrain breed in the genotype.In the present study, a significant interaction (P ≤ 0.05) was found between RYR1 genotype and carcass meatiness range in mean backfat thickness from five measurements and intramuscular fat content.Among carcasses with meatiness up to 54%, those of the pigs of CC genotype were characterised by thinner backfat than those of the pigs of CT genotype.On the other hand, fat content was higher in the pigs of CC genotype than in those of CC genotype among carcasses with meatiness above 54% (Table 6).
Important coding transcription genes play a crucial role in the development of skeletal muscles in which their expression occurs, and they belong to the MyoD family (Te Pas and Visscher 1994).In the present study, the effect of one gene of the MyoD family was analysed, i.e. the porcine myogenin (MYOG) gene, on carcass slaughter value and meat quality.The results did not reveal any significant differences between AA and AB genotypes at locus MYOG in carcass slaughter value, meat quality and its basic chemical composition.However, a significant interaction (P 0.05) was found between MYOG genotype and carcass meatiness range for water-soluble protein content (Table 7).Among carcasses with meatiness above 54%, meat of the pigs of AB genotype was characterised by higher water-soluble protein content in relation to those of AA genotype.In the study of Kapelański et al. (2005) on the stress susceptibility-gene (T)-free pigs, a higher colour lightness and higher water-soluble protein and ash contents were found in meat of the pigs of AB genotype than of AA genotype at locus MYOG.This study also shows that animals of BB genotype were characterised by more favourable meat quality in relation to those of AA genotype in locus MYOG based on water-holding capacity, free drip, water-soluble protein content and water-holding capacity sensory evaluation.However, Kapelański et al. (2004) did not show any association of MYOG gene with most analysed meat quality traits in pigs after Pietrain boars, except for pH 1 and meat colour.They found that the meat of the porkers of BB genotype was characterised by higher pH 1 and darker colour than in those of AB genotype.Also in the study of Krzęcio et al. (2007b) on the stress susceptibility-gene (T)-free pigs with respect to the quality of meat of the LD muscle, a significant association was found of MYOG genotype with pH measured after 48 h post mortem, electrical conductance after 35 min, 3 h and 24 h post mortem and dry matter content in meat.They observed lower values of electrical conductance and dry matter content in meat of the pigs of BB genotype in relation to those of AB genotype.Furthermore, in the study on the same porkers carried out by Krzęcio et al. (2007a) a higher loin weight was determined by BB genotype than that of ham by AB genotype at locus MYOG.Also Cieślak et al. (2002) found within the TORHYB programme an association of MYOG genotype with backfat thickness from several measurement points in pigs from Pietrain boars, which was thinner in animals of AB genotype than in those of BB genotype.
To recapitulate, the results lead to the conclusion that better pork quality of the CC genotype was achieved compared to that of CT genotype at locus RYR1, with similar carcass slaughter value and meat chemical composition, which confirms the need for further selection towards the elimination of the T allele from the parent herds.No significant differences were found in carcass slaughter value and meat quality traits between AA and AB genotypes at locus MYOG.No significant differences were also found between animals with lower and higher percentage of carcass meat content with respect to meat quality traits and its chemical composition, except for water-holding capacity (WHC) which was higher in pigs with meatiness ≤ 54% than in those with meatiness > 54%.Furthermore, interaction was found between carcass meatiness range and RYR1 genotype in the case of backfat thickness and intramuscular fat content, as well as between carcass meatiness range and MYOG genotype in relation to water-soluble protein content.

Table 1 .
Frequency of RYR1 and MYOG genotypes and allele in pigs

Table 2 .
The LSQ means and their standard deviations (SD) of analyzed traits and relationship between genotypes at the loci MYOG and RYR1 and meatiness for slaughter value of carcass and basic chemical composition of meat in pigs * -significant at p ≤ 0.05; ** -significant at p ≤ 0.01; ns -not significant

Table 3 .
The LSQ means and their standard deviations (SD) of analyzed traits and relationship between genotypes at the loci MYOG and RYR1 and meatiness for meat quality traits in pigs

Table 5 .
The LSQ means and standard deviation (SD) of meat quality traits depending on RYR1 genotype a,b Mean values in rows marked by different letters differ significantly at p ≤ 0.05 A,B Mean values in rows marked by different letters differ significantly at p ≤ 0.01 b ± 1.12 Results in the table are given as LSQ mean and standard deviation.a,b,c Mean values in rows marked by different letters differ significantly at p ≤ 0.05

Table 4 .
The LSQ means and standard deviation (SD) of meat quality traits depending on meatiness level

Table 7 .
Interaction (genotype MYOG × carcass meatiness) Results in the table are given as LSQ mean and standard deviation.a,b Mean values in rows marked by different letters differ significantly at p ≤ 0.05