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1 Meat Science 84 (2010) Contents lists available at ScienceDirect Meat Science journal homepage: The association between polymorphism of PKM2 gene and glycolytic potential and pork meat quality H. Sieczkowska a, M. Koćwin-Podsiadła a, *, A. Zybert a, E. Krzęcio a, K. Antosik a, S. Kamiński b, E. Wójcik b a University of Podlasie, Chair of Pig Breeding and Meat Science, 14 Prusa Street, Siedlce, Poland b University of Warmia and Mazury, Department of Animal Genetics, 5 Oczapowski Street, Olsztyn, Poland article info abstract Article history: Received 25 July 2008 Received in revised form 12 February 2009 Accepted 21 August 2009 Keywords: Pigs PKM2 gene Glycolytic potential Breed Meat quality traits The objective of this study was to investigate the association of PKM2 gene with glycolytic potential and meat quality traits in three groups of fatteners Landrace, Landrace Yorkshire and (Landrace Yorkshire) Duroc. The present study was conducted on 243 fatteners, free of RYR1 T gene, which 95 were of Landrace breed and the rest were the following crosses: 66 Landrace Yorkshire and 82 (Landrace Yorkshire) Duroc. It has been stated, that PKM2 gene (independently from the breed) was significantly associated with GP, lactate content, R 1 indicator, ph and drip loss. The presence of TT genotype may lead to increase of GP and lactate content and results in low ph 24 and ph 144 and bigger drip loss measured 96 and 144 h after the slaughter. Except for the landrace fatteners, the association of the PKM2 gene with the glycogen content has not been statistically confirmed. Statistically confirmed interaction shows, that the association of PKM2 gene with glycolytic potential and glycogen content concerns mainly the Landrace pigs. Moreover, a high (almost 89%) conformability of the genotype of PKM2 gene with the RN phenotype, can serve as an additional argument in favour of the thesis. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction * Corresponding author. Tel.: ; fax: address: kocwin@ap.siedlce.pl (M. Koćwin-Podsiadła). Changes of the basic characteristics of meat quality which occur in the tissue after the slaughter have a considerable impact on its further use in food processing. One of the reasons for these changes (apart from the slaughter method or the treatment of the carcasses after the slaughter) is metabolism of the carbohydrates in the muscle tissue, the muscle glycogen in particular. Postmortem anaerobic glycolysis leads to the accumulation of the lactate in the muscles. If lactate level is too high, pale, soft exudative (PSE), or acid meat can result. The development of PSE meat is caused by the causative mutation in skeletal muscle ryanodine receptor (RYR1) gene. The RYR1 mutation has a large impact on meat quality. In general, pigs homozygous (TT) and heterozygous (CT) for the RYR1 gene compared with non-carriers (CC) have a lower water holding capacity (WHC), higher drip loss, pale colour, and they also have accelerated postmortem glycolysis, which results in low ph during early post mortem stage (Koćwin-Podsiadła, Przybylski, Kurył, Talmant, & Monin, 1995; Rosenvold & Andersen, 2003; Sellier & Monin, 1994). Recently, there has been a search for genes responsible for the variability of glycogen content in muscle tissue, which determines the degree of glycolytic potential. One of these genes to have a connection with glycogen content in muscle tissue is PRKAG3 (protein kinase, adenosine monophosphate activated, gamma 3 subunit). The hypothesis of the existence of the PRKAG3 gene was announced by Milan et al. (2000). These researches stated that non-conservative substitution (R200Q) in the PRKAG3 gene in Hampshire pigs or crossbreed pigs including Hampshire leads to a 70% increase (in vivo) in muscle glycogen content in RN homozygous and heterozygous pigs, decrease of ph 24 and WHC. Later research conducted by Andersson (2003) and Koćwin-Podsiadła, Krzęcio, Zybert, Sieczkowska, and Antosik (2006) proved that polymorphism of PRKAG3 gene in not sufficient to diagnose all pigs with high glycolytic potential and therefore results in great losses during meat processing. Moreover, in Koćwin-Podsiadła et al. (2006) research, very low (only 7%) conformability of polymorphism of PRKAG3 gene with RN phenotype has been found. Among animals with GG (rn + rn + ) genotype in relation to PRKAG3 gene more than half (55.14%) were pigs with RN phenotype. The need to continue studies in search of other genetic conditions of glycogen content in the muscle tissue (apart from PRKAG3 gene) was also emphasised by Meadus, MacInnis, Dugan, and Aalhaus (2002), Josell et al. (2003) and Fontanesi, Davoli, Nanni Costa, Sotti, and Russo (2003). The findings mentioned above show the importance of further research to find the reasons for the variability of glycogen content in muscle tissue. The interest in genes which code enzymes taking part in the process of glycolysis has also proved to be justified. One /$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi: /j.meatsci

2 H. Sieczkowska et al. / Meat Science 84 (2010) of such enzymes is pyruvate kinase which in glycolytic pathway catalyzes the conversion of phosphenolopyruvate into pyruvate which is later reduced to lactate in anaerobic conditions (Pösö & Puolanne, 2005). Pyruvate kinase has four different isoforms: M1, M2, L and R. M1 and M2 forms can be observed in muscles, heart and brain, whereas L and R forms can be found in liver and erythrocytes (Takegawa, Shinohara, & Miwa, 1984). M1 and M2 forms are coded by PKM (Pyruvate Kinase Muscle) gene (Noguchi, Inoue, & Tanaka, 1986) which is located on porcine chromosome 7 in q12 q23 area (Davoli et al., 2002; Fontanesi et al., 2003). Fontanesi et al. (2003) found the association between polymorphism of PKM2 gene with glycogen content. Moreover, confirmation of the association between polymorphism of PKM2 gene with glycogen content could be useful to include this DNA polymorphism in marker assisted selection in pig breeding programs. In this investigations the polymorphism of PKM2 gene was tested for association with glycolytic potential and related meat quality traits in three different groups of fatteners Landrace, Landrace Yorkshire and (Landrace Yorkshire) Duroc. 2. Material and methods 2.1. Materials The present study was conducted on 243 fatteners, which 95 were of Landrace (L) breed and the rest were the following crosses: 66 Landrace Yorkshire (L Y) and 82 (Landrace Yorkshire) Duroc ((L Y) D), with a similar number of castrates and gilts. All the animals were free of RYR1 T gene. The animals came from the Jagodne Breeding Center. The maintenance and nutrition conditions were the same for all animals throughout rearing (complete feeds, fed according to age). The animals were slaughtered at kg live weight during two autumn and winter months (November and December), about animals per week with the aim of eliminating the influence slaughter season on meat quality traits. Pigs were slaughtered within 2 4 h after transport (300 km) using an electric stunner (MIDAS, Stork RMS, the Netherlands and INARCO constant voltage system) and the bled lying down, in accordance with the technology applied at the Sokołów Meat Plant, belonging to SOKOŁÓW S.A. in Sokołów Podlaski Methods The quality of fresh (up to 45 min immediately after bleeding) and cooled (after 24 h chilling) meat was evaluated after slaughter on the m. Longissimus lumborum (LL) (after last rib), on the basis of the following parameters: acidity of the muscle tissue (ph) measured directly in the LL muscle 35 min, 24 h, 48 h, 96 h and 144 h postmortem (ph 35, ph 24,pH 48,pH 96,pH 144, respectively), using a pistol ph-meter MASTER (Draminski, Olsztyn, Poland) calibrated with temperature compensation; electrical conductivity (EC) measured with a LF-Star conductometer (Ingenieurbüro Matthäus, Noblitz, Germany) 2 h and 24 h postmortem (EC 2,EC 24 ); colour lightness (L * ) of the muscle tissue, was assessed 24 h postmortem with a Minolta portable chroma meter (model CR 310, Minolta, Osaka, Japan) using D65 illuminant and 50 mm orifice; rate of ATP breakdown, expressed by R 1 = IMP/ATP indicator, determined 45 min postmortem on meat samples taken after last rib, according to the method of Honikel and Fischer (1977); WHC, determined by the filter paper method according to the method of Grau and Hamm (1952) as modified by Pohja and Ninivaara (1957), 24 h postmortem; drip loss, determined according to Prange, Juggrt, and Scharner (1977), 48 h, 96 h, and 144 h postmortem; meat yield in the curing and thermal processing (72 C), expressed by TY (technological yield) indicator, according to Naveau, Pommeret, and Lechaux (1985) as modified by Koćwin-Podsiadła et al. (2004). The samples of LL muscle were taken 24 h after slaughter. Meat cubes cm were immersed in a solution containing of 12% NaCl, 0.07% NaNO 2 and 0.06% of glucose. After 24 h of curing in 4 C the samples were thermal processed in a water bath (to an internal temperature of 72 C). The water, dry matter, total protein and intramuscular fat content (IMF) of LL muscle were determined in accordance with following procedures recommended by the AOAC: water and dry matter procedure No , protein , and IMF (AOAC, 2000). The samples cut from LL muscle 45 min postmortem (immediately immersed into tubes with 1 M HClO 4 and homogenized to inhibit glycolytic changes) were analyzed for the glycolytic potential (GP) and content of glycogen and lactate. The content of glycogen was determined by the enzymatic method according to Darymple and Hamm (1973) and lactate content according to Bergmeyer (1974). The glycolytic potential was calculated as the sum of: 2[glycogen] + [lactate] according to Monin and Sellier (1985) and expressed as lmol of lactic acid equivalent per g of fresh muscle. The genomic DNA was isolated from white blood cells according to Kawasaki (1990) and Coppieters, van Zeveren, van de Weghe, Peelman, and Bouquet (1992). The RYR1 C1843T polymorphic site was analyzed with DNA test using the PCR/RFLP method, according to Fujii et al. (1991). The mutation in the 3 0 -untranslated region was investigated using PCR SSCP method (Fig. 1), according to Fontanesi et al. (2003) with own modification. PCR was performed in total volume 10 ll using 240 ng porcine DNA, 0.32 ll each primer (forward: 5 0 AGGCGGCTGCAGTAGTCG 3 0, reverse: 5 0 CCCCTTAGCC TCCCTCACTC 3 0 ), 0.52 ll dntp mix (2 mm), 0.20 ll 25 mm MgCl 2, 1.60 ll enhancer (Epcentre, Madison, WI, USA), 0.60 ll20 PCR buffer, 1 U/llTfl polymerase (Epicentre, Madison, WI, USA) 5.72 ll water. Amplification was carried out using MJ Research Thermal Cycler (MJ Research Inc., USA). After an initial denaturation step of 3 min at 95 C, 35 cycles were performed as follows: 25 s at 95 C, 25 s at 61 C and 25 s at 72 C. A final extension step at 72 C for 5 min followed the PCR cycles. Aliquots (2.5 ll) of each PCR product were added to 5 ll denaturating buffer (50 mm NaOH and 1 mm EDTA), denaturated at 85 C for 13 min and electrophoresed in 10% polyacrylamide gel (ETC, Elektrophorese-Technik, Germany) in 12.5 C as follows: preelectrophoresis (200 V, 20 ma, 10 W) for 10 min, electrophoresis I (200 V, 20 ma, 10 W) for 10 min and II (375 V, 30 ma, 15 W) for 2 h. Single standard DNA conformers were revealed by silver staining (Promega, Madison, WI, USA). Associations between the genotypes of PKM2 gene and meat quality traits (glycogen, lactate content, GP, ph, R 1, EC, L *, WHC, drip loss and TY) were assessed using two way analysis of variance Fig. 1. The PCR SSCP at PKM2 locus. The genotypes of the corresponding gel lines are indicated at the top of a figure.

3 182 H. Sieczkowska et al. / Meat Science 84 (2010) in non-orthogonal scheme in Statistica 6.0 (StatSoft, Tulsa, OK, USA) with following model: y ij ¼ l þ a i þ b j þ ab ij þ e ijk where: l the overall mean; a i the effect of PKM2 genotype, i =1, 2, 3; b j the effect of breed or crossbreed, j =1,2,3;ab ij the interaction between PKM2 genotype and breed (crossbreed); e ijk the random residual. In this investigation, the additional one way analysis of variance in group of pure breed (L) fatteners was performed using following model: Y i ¼ l þ a i þ e ij where: l the overall mean; a i the effect of PKM2 genotype, i =1, 2, 3; e ij the random residual. All the mean values were compared using Tukey s test. 3. Results and discussion Table 1 shows the frequency of alleles and the observed and expected frequency of genotypes. The data obtained proves that the L and (L Y) D populations were genetically balanced. L Y population was in Hardy Weinberg disequilibrium. Table 2 reports the effect of breed, PKM2 gene and interaction between analyzed breeds and genotypes of PKM2 gene for investigated in our work meat quality traits. It was found, that homozygous TT fatteners of the PKM2 gene in comparison with animals with CC genotype were characterized by the biggest difference in glycolytic potential (Table 3), however, the difference in standard deviation (SD) units (between TT and CC fatteners) was rather low and amounted about 0.45 SD. The association between polymorphic forms of the PKM2 gene and lactate content has also been confirmed. The higher lactate content was detected in postmortem muscles of TT genotype compared with CT and CC (higher for 5.97 and 5.09 lmol/g respectively). In contrast to Fontanesi et al. (2003), our work did not prove a significant association between the polymorphism of the PKM2 gene, and glycogen content (Table 2 and Table 3). In the present study, we consider the PKM2 gene as the candidate for glycolytic potential and glycogen content in porcine skeletal muscles. The negative effect of RN - phenotype on these parameters is most obvious. In case of RN - phenotype, defined on the basis of glycolytic potential, the effect is so strong that the difference between RN - carriers and non-carriers significantly exceeds one SD unit. The carriers of RN - gene compared with non-carriers have also 40 70% higher muscle glycogen content (Larzul, Le Roy, Monin, & Sellier, 1998; Lundstrom, Andersson, Maerz, & Hansson, 1994; Przybylski, Koćwin-Podsiadła, Kurył & Monin, 1996). However the influence of RN - phenotype on the lactate content has not been ascertained (Lundstrom et al., 1994; ð1þ ð2þ Table 2 The influence of breed, PKM2 genotype and their interaction for quality traits of LL muscle. Trait F-emp./p value GP (lmol/g) 2.63 Glycogen (lmol/g) 1.07 Lactate (lmol/g) 2.88 ph R ph ph ph ph EC 2 (ms/cm) 1.35 EC 24 (ms/cm) 2.04 L * 0.64 Drip loss 48 h (%) 0.93 Drip loss 96 h (%) 3.51 Drip loss 144 h (%) 2.99 WHC (cm 2 ) 0.25 TY (%) 1.23 Protein content (%) 1.95 IMF content (%) 0.38 Water content (%) 0.63 NS Dry matter content (%) 0.87 Gene Breed Interaction Gene breed NS NS 0.27 GP glycolytic potential, EC electrical conductivity, L * meat lightness, WHC water holding capacity, TY technological yield, IMF intramuscular fat content; NS not statistically confirmed. Przybylski et al., 1996). From numerous studies it can be concluded that a high glycolytic potential in muscles, results in low ultimate ph, pale colour, and a high drip loss (Larzul et al., 1998; Lundstrom et al., 1994; Przybylski et al., 1996; Sellier, 1998). Table 1 The frequency of alleles and genotypes of PKM2 gene. Breed PKM2 Frequency alleles Frequency of genotypes HWE a p-value L n =95 L Y n =66 (L Y) D n =82 Observed Expected CC CT TT C T CC CT TT CC CT TT NS NS ** NS not statistically significant. a HWE Hardy Weinberg equilibrium. ** Significance at p (significance indicate a discrepancy from HWE).

4 H. Sieczkowska et al. / Meat Science 84 (2010) Table 3 The effect of PKM2 genotype and breed on quality traits of LL muscle. Trait Genotype Breed Total CC n =87 CT n = 123 TT n =33 L n =95 L Y n =66 (L Y) D n =82 GP (lmol/g) a ± b ± b ± b ± a ± a ± ± Glycogen (lmol/g) ± ± ± b ± ab ± a ± ± Lactate (lmol/g) 37.72a ± a ± b ± b ± a ± b ± ± R a ± b ± b ± B ± B ± A ± ± 0.04 ph b ± a ± a ± A ± A ± B ± ± 0.13 ph ± ± ± A ± A ± B ± ± 0.12 ph ± ± ± a ± a ± b ± ± 0.10 ph b ± a ± a ± A ± A ± B ± ± 0.13 EC 24 (ms/cm) 3.91 ± ± ± A ± A ± B ± ± 1.16 Drip loss 48 h (%) 6.11 ± ± ± B ± B ± A ± ± 2.34 Drip loss 96 h (%) 9.17a ± b ± b ± B ± B ± A ± ± 2.95 Drip loss 144 h (%) 11.46a ± ab ± b ± B ± B ± A ± ± 2.83 IMF content (%) 1.77 ± ± ± A ± A ± B ± ± 0.67 Dry matter content (%) ± ± ± A ± A ± B ± ± 1.02 GP glycolytic potential, EC electrical conductivity, IMF intramuscular fat content; means signed by different capital letters A, B differs statistically at p ; means signed by different small letters a, b differs statistically at p In the present study has been found, that TT animals in relation to PKM2 gene compared to CC pigs had accelerated energy consumption (higher value of R 1 indicator), lower ph 24 and ph 144 and increased drip loss measured 96 and 144 h after the slaughter (Table 3). It should be mentioned, that in relation to the parameters of the meat quality, which were analyzed in this work and statistically confirmed, the animals identified as CT and TT of the examined gene except the lactate content create a homogeneous group. Glycolytic potential (umol/g) Glycogen (umol/g) ab c ab c ab ab ab a a 47.74ab 45.70a 43.31a CC CT TT PKM2 genotype L LxY (LxY)xD 53.79b 47.35ab 43.33a CC CT TT PKM2 genotype L LxY (LxY)xD 50.29b 46.44ab 37.91a Fig. 2. The interaction between PKM2 gene and breed for glycolytic potential and glycogen content. Means signed by different small letters a, b, c differs statistically at p The analysis of the effect of the second factor the breed (independently from the genotype of the PKM2 gene), showed that the pure breed (L) was characterized by a higher glycolytic potential ( lmol/g: p ) then either of the crossbreeds (L Y: ; (L Y) D lmol/g: p ). The L fatteners compared with L Y and (L Y) D crossbreeds had also a higher glycogen content (L: 50.48; L Y: 47.19; (L Y) D: lmol/g: p ). It has also been observed, that L fatteners in comparison with L Y crossbreeds had a higher lactate content, however, it was comparable to the value of this parameter obtained in the group of (L Y) D(Table 3). Meat from the pure breed (L) pigs, compared with (L Y) D crossbreeds, was also found to have accelerated energetic consumption (a higher value of R 1 indicator), lower ph (measured at 24, 48 and 144 h after the slaughter) and significantly (p ) higher drip loss measured 48, 96 and 144 h postmortem. Moreover, pure breed (L) fatteners had also lower IMF and dry matter content then (L Y) D crossbreeds (Table 3). However, it should be noticed that regarding the param- Table 4 The influence of PKM 2 genotype on meat quality traits within pure L breed. Trait PKM2 Gene F-emp. p-value GP (lmol/g) Glycogen (lmol/g) Lactate (lmol/g) ph NS R ph NS ph NS ph ph EC 2 (ms/cm) 1.97 NS EC 24 (ms/cm) 1.44 NS L* Drip loss 48 h (%) 1.18 NS Drip loss 96 h (%) Drip loss 144 h (%) 2.03 NS WHC (cm 2 ) 2.31 NS TY (%) 0.64 NS Protein content (%) 0.60 NS IMF content (%) 0.75 NS Water content (%) 0.50 NS Dry matter content (%) 0.00 NS GP glycolytic potential, EC electrical conductivity, L* meat lightness, WHC water holding capacity, TY technological yield, IMF intramuscular fat content; NS not statistically confirmed.

5 184 H. Sieczkowska et al. / Meat Science 84 (2010) Table 5 Association analysis between PKM2 genotype and meat quality parameters within pure L breed. Trait PKM2 genotype Total CC n =22 CT n =49 GP (lmol/g) a ± b ± b ± ± Glycogen (lmol/g) 43.31a ± b ± b ± ± Lactate (lmol/g) 37.20a ± ab ± b ± ± R a ± ab ± b ± ± 0.03 ph b ± a ± a ± ± 0.08 ph b ± a ± a ± ± 0.11 L* 53.49a ± ab ± b ± ± 3.24 Drip loss 96 h (%) 9.79a ± b ± b ± ± 2.92 Means signed by different small letters a, b differs statistically at p ; GP glycolytic potential, L* meat lightness. TT n =24 eters of the meat quality discussed above and statistically confirmed (except the lactate and glycogen content), pure breed L fatteners and the L Y crossbreeds were homogeneous. The interaction between PKM2 gene and the genetic group confirmed in this study for glycolytic potential and the glycogen content indicates that the association between the polymorphism of PKM2 gene with the meat quality concerns mainly the Landrace fatteners (Fig. 2 and Table 3). The results of interaction between both analyzed main factors confirmed statistically for glycolytic potential and the glycogen content induced us to additional analysis of the variance concerning the effect of PKM2 gene on the meat quality traits in pure breed (L) fatteners. (Tables 4 and 5). It was found, that L CT and TT animals, in relation to the PKM2 gene, in comparison with CC pigs were characterized by significantly higher (p ) glycolytic potential, (for about 21 lmol/ g) and glycogen content, while L pigs with TT genotype of PKM2 gene compared with fatteners of CC genotype had a higher lactate content (Table 5). It should be also mentioned that the effect of the PKM2 gene on glycogen content and the glycolytic potential, in discussed above group of Landrace fatteners is not so strong as in case of RN - phenotype. The differences in SD units (between CC and TT pigs) were lower then 1 SD and amounted 0.64 and 0.43 SD (respectively for the glycolytic potential and the glycogen content). However it ought to be stressed that despite weak effect (in SD units) of PKM2 gene on the glycolytic potential and glycogen content, the impact PKM2 gene is similar to the RN - phenotype. The analysis of conformability between polymorphic forms of the PKM2 gene with RN - phenotype conducted on a group of L fatteners showed its high, almost 89%, conformability with RN - phenotype (CT and TT genotypes in relation to PKM2 gene vs. carriers of RN unpublished data). The statistic analysis of the meat quality traits according to polymorphic forms of the PKM2 gene conducted on L fatteners, proved that the meat from TT animals in comparison with CC pigs were characterized by significantly lower (p ) ph 96 and ph 144, higher meat lightness and drip loss measured in 96 h postmortem (Table 5). 4. Conclusions It has been stated, that PKM2 gene (independently from the breed) was significantly associated with GP, lactate content, R 1 indicator, ph and drip loss. The presence of TT genotype may lead to increase of GP and lactate content and results in low ph 24 and ph 144 and bigger drip loss measured 96 and 144 h after the slaughter. We also showed, that pure breed L fatteners in contrast to (L Y) D crossbreeds were characterized by a higher GP and lactate content, lower ph (at 24, 48 and 144 h after the slaughter) and significantly (p ) higher drip loss measured 48, 96 and 144 h postmortem. 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