Relationship between the Porcine Stress Syndrome gene and pork quality traits of F2 pigs resulting from divergent crosses
http://www.100md.com
《遗传学和分子生物学》
IUniversidade Federal de Viosa, Departamento de Zootecnia, Viosa, MG, 36571-000, Brazil
IIUniversidade Federal de Viosa, Departamento de Tecnologia de Alimentos, Viosa, MG, Brazil
ABSTRACT
The PSS genotypes of 596 F2 pigs produced by initial mating of Brazilian commercial sows and native boars were characterized by PCR-RFLP and the pork quality traits were evaluated. Among the 596 pigs studied, 493 (82.7%) were NN and 103 (17.3%) were Nn. There were no differences between NN and Nn pigs in the following pork qualities: pHu (5.71 ± 0.16 vs 5.70 ± 0.11), intramuscular fat (1.55 ± 0.64% vs 1.65 ± 0.67%), shear force (5552 ± 878 g/1.2 cm vs 5507 ± 826 g/1.2 cm), lightness (44.96 ± 2.05 vs 45.01 ± 1.92), redness (0.64 ± 0.60 vs 0.79 ± 0.55), yellowness (6.62 ± 0.56 vs 6.65 ± 0.48), hue (84.28 ± 5.53 vs 83.41 ± 4.85), or chroma (6.68 ± 0.52 vs 6.73 ± 0.52). However, pork from Nn pigs had a significantly (p < 0.05) lower pH45 (6.41 ± 0.27 vs 6.51 ± 0.26) and greater drip (3.92 ± 1.90% vs 3.06 ± 1.60%), cooking (33.29 ± 2.26% vs 32.50 ± 2.54%) and total (35.67 ± 2.48% vs 34.01 ± 2.58%) loss compared to that of NN pigs. These results indicate that, even in divergent crosses, PSS gene carriers produce pork of poorer quality.
Key words: meat quality, PCR-RFLP, pork, PSE, PSS, pig.
Introduction
Meat is one of the main sources of protein in the human diet, and pork is one of the most produced and consumed worldwide (Franco et al., 1998). One of the concerns during pork production is its quality. Historically, considerations about pork quality have been restricted to aspects related to health, processing, nutrition and, to a lesser extent, sensory traits. However, in recent years, pork consumers have become increasingly concerned about the safety of this meat, its ease of preparation, and their satisfaction during consumption. Consumer satisfaction during pork consumption is related to sensory perception of attributes such as meat color, juiciness and tenderness, which are adversely affected by the development of the PSE (pale, soft and exudative) condition after death. One of the main economic losses in the pig industry is related to PSE pork, which originates from animal stress and depends upon pig genetics and animal handling conditions before and during slaughtering. Stress conditions can activate malignant hyperthermia in pigs homozygous for the PSS (Porcine Stress Syndrome) gene (nn) and may even cause death (Fisher et al., 2000a). Since PSS gene carriers have a higher probability of presenting poor quality pork (Santana et al., 1998), there is a constant concern about pig welfare during handling and transport before slaughter (Geers et al., 1994).
Recent advances in our understanding of the regulation of skeletal muscle contraction has led to the identification of a mutation in the ryanodine receptor in the sarcoplasmic reticulum calcium release channel that has been correlated with PSS and malignant hyperthermia. Stress-susceptible pigs respond to halothane anesthesia with limb muscle rigidity, increased anerobic metabolism and increased body temperature (Rempel et al., 1993). The increased anerobic metabolism in muscle produces a sudden decrease in pH after death (pH < 5.5 24 h after slaughter) that, together with an increase in muscle temperature, leads to protein denaturation and adversely affects pork quality traits such as color and water-holding capacity and results in excessive water loss during pork preparation (Favero, 2002).
Theoretically, at the same finishing level (same fat depth), heavier pigs should show a greater tendency to develop PSE because heavier carcasses take longer to cool because of their larger volume/surface area ratio. This is a problem directly related to the muscles of the ham that cool more slowly than muscles such as the longissimus dorsi. Consequently, heavier pigs show a greater tendency to develop PSE pork. Moreover, the muscle glycogen content is higher in heavier pigs, leading to rapid postmortem increases in glycolysis and, consequently, greater decreases in muscle pH after slaughter (Ellis and Bertol, 2001).
A high water-holding capacity of pork used to manufacture hams and sausages has a direct impact on the quality of these processed products because it reduces drip loss in fresh and frozen products, and also reduces cooking loss and maintains the product's juiciness. Substances such as phosphates and rusk increase the water-holding capacity and are used to produce hams and sausages from PSE pork. However, Fisher et al. (2000c) have shown that the addition of phosphate does not increase pork water absorption, but causes pork products to bind water more completely, thereby minimizing losses during processing.
Genetic factors are among the traits that affect pork quality, and the identification of major genes and molecular markers is a promising approach for improving economic traits, such as pork quality, that are not measurable in breeding animals (Favero, 2002).
Franco et al. (1998) demonstrated that the presence of the n allele led to higher pork drip loss in the semimembranous muscle, thereby reducing its quality. Lundstrom et al. (1995) showed that the PSS gene affected the quality of the longissimus dorsi and resulted in a less tender, less desirable pork. According to Fisher et al. (2000a), a lower initial pH (within 45 min) in Nn pigs, and especially in nn pigs, compromises pork quality because of rapid glycolysis after slaughter.
The use of genomic markers to help in the selection of pork quality is one of the most promising developments in the pig industry. PSE has been known for some time to be associated with variations in the recessive PSS gene (Plastow, 2000), and the availability of a test based on the identification of the causative mutation in the PSS gene described by Fujii et al. (1991) was a key step in marker-assisted screening of pork quality. The test allows breeders to accurately separate all three PSS genotypes, instead of just reactors (nn) from non-reactors (NN and Nn), and has allowed more detailed studies of the effect of this mutation on pork quality.
The aim of this study was to determine the PSS genotypes in F2 pigs derived from divergent crosses and to determine their relationship to pork quality traits.
Materials and Methods
The 596 genotyped F2 pigs were produced by outbreed crossing using 18 commercial females (11 Landrace x Large White and seven Landrace x Large White x Pietrain) with two Brazilian native boars (Piau breed). Both boars and 11 parental females had the NN genotype. The F2 pigs were reared and slaughtered on the Pig Breeding Farm maintained by the Department of Animal Science, Universidade Federal de Viosa, Viosa, Minas Gerais State, Brazil. The pigs were slaughtered at a live weight of 65.0 ± 5.5 kg and were deprived of food for 18 h before slaughter, but had access to fresh water ad libitum. The pigs were electrically stunned (300V/5 s) and bled by cardiac puncture under the left armpit.
For each pig, the pH (pH45) of the longissimus dorsi was measured 45 min postmortem in the left half of the carcass before cooling and in the right half of the carcass after cooling at 4 °C for 24 h in horizontal freezers. Samples of the longissimus dorsi were then obtained to measure other pork quality traits.
Pork quality traits were evaluated in the Meat Laboratory of the Department of Food Technology, Universidade Federal de Viosa, using the procedures described by Benevenuto Junior (2001) for pH 24 h postmortem (pHu), intramuscular fat (IMF), drip, cooking and total loss, shear force, and objective meat color (lightness, redness, yellowness, hue and chroma).
Genotypic analysis was done in the Laboratory of Animal Biotechnology of the Department of Animal Science, Universidade Federal de Viosa. DNA was salt-extracted from white blood cells collected immediately after slaughter using a standard laboratory protocol. The sequence of the ryr-1 gene that contains the C T mutation responsible for triggering PSS (Fujii et al., 1991) was amplified by PCR-RFLP using the primers cited by O'Brien et al. (1993) and generated a 659 bp product.
The amplification mixture contained 1 U of Taq DNA polymerase (Phoneutria), 0.2 M of each primer (forward - 5'-TCCAGTTTGCCACAGGTCCTACCA-3' - and reverse - 5'-TTCACCGGAGTGGAGTCTCTGAG-T-3'), 2 mM MgCl2, 20 mM Tris, pH 8.3, 50 mM KCl, 0.2 mM dNTPs, and 25 ng of genomic DNA in a final volume of 20 mL, according to the standard protocol described by Fujii et al. (1991).
The samples were distributed into previously labeled microtubes containing the reagent mixture described above and were centrifuged at 7,826 g for 10 s to ensure that the samples were at the bottom of each tube. The microtubes were then placed in the 96-sample tray of the thermocycler (MJ-Research PTC-100). The amplification program, modified from Fujii et al. (1991) and Houde et al. (1993), consisted of initial denaturation at 94 °C for 3 min and 35 cycles at 94 °C for 45 s, 68 °C for 1 min and 72 °C for 1 min, with a final polymerization step at 72 °C for 5 min.
Mutation analysis of the samples amplified as described above was done using the restriction enzyme BsiHKA I (New England Biolabs). This enzyme cleaves the 659-bp sequence containing the PSS mutation and generates fragments of 524 and 135 bp in normal homozygotes (NN), fragments of 524, 358, 166 and 135 bp in heterozygotes (Nn), and fragments of 358, 166 and 135 bp in mutant homozygous pigs (nn). After digestion, the reaction products were analyzed on 8% silver nitrate-stained polyacrylamide gels and the pigs were classified as normal homozygotes (NN), heterozygotes (Nn) and recessive homozygotes (nn) according to the size of the DNA fragments.
Statistical analysis of the association of the genotypes with the traits evaluated was done using the SAS General Linear Models (SAS, 1997) program, according to the following model:
Yijkl = m + Gi + Sj + Lk + eijkl
where Yijkl = observed trait in an animal of genotype i, sex j and batch k, m = general mean, Gi = genotype effect (NN or Nn), Sj = sex effect (1 = castrated male and 2 = female), Lk = batch effect (k = 1, 2, 3, 4 and 5) and eijkl = random error.
Results
The RFLP patterns were as expected. Pigs homozygous for the mutation (nn) were characterized by fragments of 358, 166 and 135 bp, normal pigs (NN) showed the 524-bp and the complementary 135-bp fragment, and heterozygous pigs showed fragments of 524, 358, 166 and 135 bp. The NN and Nn genotypes were found in 493 (82.7%) and 103 (17.3%) pigs, respectively. Since only one nn pig was identified, it was not considered in the analysis. These unusual frequencies in F2 crosses were found to be the result of divergent matting patterns in which parental boars were not carriers and the F1 generation was randomly mated regardless of the PSS genotype.
The mean results and number of observations for each pork quality trait in each genotype are shown in Table 1. The traits showing significant differences between the NN and Nn genotypes were pH45 and drip, cooking and total losses. Nn pigs had a higher glycolytic rate postmortem, as indicated by their lower (p < 0.05) pH45 values and higher (p < 0.05) drip, cooking and total losses.
Discussion
The significantly lower pH45 and higher drip, cooking and total losses seen in Nn pigs agreed with other reports in the literature (McPhee and Trout, 1995; Lundstrom et al., 1995; Leach et al., 1996; Monin et al., 1999; Fernandez et al., 2002; Green, 1997; Franco et al., 1998; Miller et al., 1999; Jeremiah et al., 1999; Fisher et al., 2000b) and showed that the presence of the n allele negatively affected pork quality traits by producing greater muscle acidity that led to greater losses during storage and cooking, and produced less juicy pork.
Drip loss, an indicator of the muscle's water holding capacity, is negatively correlated with pH45 (Benevenuto, 2001) and is highly dependent on the initial denaturation of pork myofibrillar proteins.
The lack of a significant difference in objective color indices and tenderness between the Nn and NN genotypes has also been observed by others (Bastos et al., 2001; Fernandez et al., 2002; Miller et al., 1999; Jeremiah et al., 1999). However, discrepancies in pork quality traits have also been reported. Differences in lightness (McPhee and Trout, 1995; Leach et al., 1996; Green, 1997; Bastos et al., 2001), pH24 (Leach et al., 1996; Green, 1997), shear force (Fisher et al., 2000a) and intramuscular fat (Zhang et al., 1992; Leach et al., 1996) between the NN and Nn genotypes have been considered to be indicative of the negative effect of the n allele on protein denaturation and pork quality (Fisher et al., 2000a). According to the latter authors, pork from Nn pigs is less tender and paler than pork from NN pigs because of higher shear force and lightness values (p < 0.05), both of which are associated with a higher incidence of PSE pork as a result of sarcoplasmic protein denaturation and a consequent reduction in pork quality.
In the present study, PSS gene carriers (Nn pigs) had a poorer pork quality, as indicated by their lower pH45 and higher drip, cooking and total losses. The effect of the PSS gene was demonstrable in pigs resulting from divergent crosses, which are generally less susceptible to stress. These results indicate that this gene is one of the main genes to be studied in relation to pork quality.
Acknowledgments
This work was supported by FAPEMIG, CNPq and CAPES.
References
Bastos RG, Federezzi J, Deschamps JC, Cardellino RA and Dellagostin OA (2001) Efeito do gene do estresse suino sobre caracteristicas de quantidade e qualidade de carcaa. Revista Brasileira de Zootecnia 30:37-40.
Benevenuto Junior AA (2001) Avaliao de rendimento de carcaa e de qualidade da carne de suinos comerciais, de raa nativa e cruzados. MSc thesis, Universidade Federal de Viosa, Viosa.
Ellis M and Bertol TM (2001) Efeitos do peso de abate sobre a qualidade de carne suina e da gordura. 2o. Internacional Virtual Conference on Pork Quality, http://www.conferencia.uncnet.br/pork (accessed on 6/12/2001).
Favero JA (2002) Carne suina de qualidade: Uma exigência do consumidor moderno. Anais do I Congresso Latino Americano de Suinocultura (Palestras):56-66. I Congresso Latino Americano de Suinocultura, Foz do Iguau, PR, Brazil.
Fernandez X, Neyraud E, Astruc T and Sante V (2002) Effects of halothane genotype and pre-slaughter treatment on pig meat quality. Part 1. Post mortem metabolism, meat quality indicators and sensory traits of m. longissimus lumborum. Meat Science 62:429-437.
Fisher P, Mellett, FD and Hoffman LC (2000a) Halothane genotype and pork quality. 1. Carcass and meat quality traits from the three halothane genotypes. Meat Science 54:97-105.
Fisher P, Mellett FD and Hoffman LC (2000b) Halothane genotype and pork quality. 2. Cured meat products from the three halothane genotypes. Meat Science 54:107-111.
Fisher P, Mellett FD and Hoffman LC (2000c) Halothane genotype and pork quality. 3. Comminuted meat products derived from the three halothane genotypes. Meat Science 54:113-117.
Franco MM, Antunes RC, Bernardes LAH, Olivo R, Franco FO, Carneiro AL, Rodrigues MC, Melo LM e Goulart LR (1998) Avaliao de qualidade de carne em 3 raas puras de suinos (Landrace, Large White e Pietrain) e suas relaes com o gene halotano. In: Silva MA (ed) Anais do II Simposio Nacional de Melhoramento Animal, Sociedade Brasileira de Melhoramento Animal, Uberaba, pp 455-456.
Fujii J, Otsu K, Zorzato F, De Leon S, Khanna VK, Weiler JE, O'Brien PJ and MacLennan DH (1991) Identification of a mutation in the porcine ryanodine receptor associated with malignant hyperthermia. Science 253:448-451.
Geers R, Bleus E and Van Schie T (1994) Transport of pigs different with respect to the halothane gene: Stress assessment. Journal of Animal Science 72:2552-2558.
Green BK (1997) The effects of genotype, chromium picolinate supplementation, sex, and their interactions on growth performance, carcass characteristics, and muscle quality in pigs. MSc thesis, Faculty of the Virginia Polytechnic Institute and State University, Virginia.
Houde A, Pommier AS and Roy R (1993) Detection of the ryanodine receptor mutation associated with malignant hyperthermia in purebred swine populations. Journal of Animal Science 71:1414-1418.
Jeremiah LE, Gibson JP, Gibson LL, Ball RO, Acker C and Fortin A (1999) The influence of breed, gender and PSS (halothane) genotype on meat quality, cooking loss and palatability of pork. Food Research International 32:59-71.
Leach LM, Ellis M, Sutton DS, McKeith FK and Wilson ER (1996) The growth performance, carcass traits, and meat quality of halothane carrier and negative pigs. Journal of Animal Science 74:934-943.
Lundstrom K, Karlsson A, Hakansson J, Hansson I, Johansson M, Andersson L and Andersson K (1995) Production, carcass, and meat quality traits of F2-breed crosses between European wild pigs and domestic pigs including halothane gene carriers. Animal Science 61:325-331.
McPhee C and Trout GR (1995) The effects of selection for lean growth, and the halothane allele on carcass and meat quality of pigs transported long and short distances for slaughter. Livestock Production Science 42:55-62.
Miller D, Ellis M and McKeith FWE (1999) Influence of sire line and halothane genotype on growth performance, carcass and meat quality traits in pigs. http://www.ansci.uiuc.edu/porknet/ (accessed on 6/4/1999).
Monin G, Larzul PR, Culioli J, Mourot J, Rousset-Akrim S, Talmant A, Touraille C and Sellier P (1999) Effects of the halothane genotype and slaughter weight on texture of pork. Journal of Animal Science 77:408-415.
O'Brien PT, Shen H, Cory CR and Zhang X (1993) Use of a DNA-based test for the mutation associated with Porcine Stress Syndrome (malignant hyperthermia) in 10,000 breeding swine. JAVMA 203:842-851.
Plastow GS (2000) Molecular genetics in the swine industry. Anais do III Simposio Nacional de Melhoramento Animal, Belo Horizonte, MG, pp 21-30.
Rempel WE, Lu M, Kandelgy SE, Kennedy CFH, Irvin LR, Mickelson JR and Louis CF (1993) Relative accuracy of the halothane challenge test and a molecular genetic test in detecting the gene for Porcine Stress Syndrome. Journal of Animal Science 71:1395-1399.
Santana BAA, Borges GSN, Franco MM, Bernardeli K, Nunes ALP, Antunes RC, Borges M and Goulart LR (1998) Aplicao da genotipagem do gene halotano ao melhoramento genetico suino. Anais... II Simposio Nacional de Melhoramento Animal, Uberaba, MG, pp 453.
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IIUniversidade Federal de Viosa, Departamento de Tecnologia de Alimentos, Viosa, MG, Brazil
ABSTRACT
The PSS genotypes of 596 F2 pigs produced by initial mating of Brazilian commercial sows and native boars were characterized by PCR-RFLP and the pork quality traits were evaluated. Among the 596 pigs studied, 493 (82.7%) were NN and 103 (17.3%) were Nn. There were no differences between NN and Nn pigs in the following pork qualities: pHu (5.71 ± 0.16 vs 5.70 ± 0.11), intramuscular fat (1.55 ± 0.64% vs 1.65 ± 0.67%), shear force (5552 ± 878 g/1.2 cm vs 5507 ± 826 g/1.2 cm), lightness (44.96 ± 2.05 vs 45.01 ± 1.92), redness (0.64 ± 0.60 vs 0.79 ± 0.55), yellowness (6.62 ± 0.56 vs 6.65 ± 0.48), hue (84.28 ± 5.53 vs 83.41 ± 4.85), or chroma (6.68 ± 0.52 vs 6.73 ± 0.52). However, pork from Nn pigs had a significantly (p < 0.05) lower pH45 (6.41 ± 0.27 vs 6.51 ± 0.26) and greater drip (3.92 ± 1.90% vs 3.06 ± 1.60%), cooking (33.29 ± 2.26% vs 32.50 ± 2.54%) and total (35.67 ± 2.48% vs 34.01 ± 2.58%) loss compared to that of NN pigs. These results indicate that, even in divergent crosses, PSS gene carriers produce pork of poorer quality.
Key words: meat quality, PCR-RFLP, pork, PSE, PSS, pig.
Introduction
Meat is one of the main sources of protein in the human diet, and pork is one of the most produced and consumed worldwide (Franco et al., 1998). One of the concerns during pork production is its quality. Historically, considerations about pork quality have been restricted to aspects related to health, processing, nutrition and, to a lesser extent, sensory traits. However, in recent years, pork consumers have become increasingly concerned about the safety of this meat, its ease of preparation, and their satisfaction during consumption. Consumer satisfaction during pork consumption is related to sensory perception of attributes such as meat color, juiciness and tenderness, which are adversely affected by the development of the PSE (pale, soft and exudative) condition after death. One of the main economic losses in the pig industry is related to PSE pork, which originates from animal stress and depends upon pig genetics and animal handling conditions before and during slaughtering. Stress conditions can activate malignant hyperthermia in pigs homozygous for the PSS (Porcine Stress Syndrome) gene (nn) and may even cause death (Fisher et al., 2000a). Since PSS gene carriers have a higher probability of presenting poor quality pork (Santana et al., 1998), there is a constant concern about pig welfare during handling and transport before slaughter (Geers et al., 1994).
Recent advances in our understanding of the regulation of skeletal muscle contraction has led to the identification of a mutation in the ryanodine receptor in the sarcoplasmic reticulum calcium release channel that has been correlated with PSS and malignant hyperthermia. Stress-susceptible pigs respond to halothane anesthesia with limb muscle rigidity, increased anerobic metabolism and increased body temperature (Rempel et al., 1993). The increased anerobic metabolism in muscle produces a sudden decrease in pH after death (pH < 5.5 24 h after slaughter) that, together with an increase in muscle temperature, leads to protein denaturation and adversely affects pork quality traits such as color and water-holding capacity and results in excessive water loss during pork preparation (Favero, 2002).
Theoretically, at the same finishing level (same fat depth), heavier pigs should show a greater tendency to develop PSE because heavier carcasses take longer to cool because of their larger volume/surface area ratio. This is a problem directly related to the muscles of the ham that cool more slowly than muscles such as the longissimus dorsi. Consequently, heavier pigs show a greater tendency to develop PSE pork. Moreover, the muscle glycogen content is higher in heavier pigs, leading to rapid postmortem increases in glycolysis and, consequently, greater decreases in muscle pH after slaughter (Ellis and Bertol, 2001).
A high water-holding capacity of pork used to manufacture hams and sausages has a direct impact on the quality of these processed products because it reduces drip loss in fresh and frozen products, and also reduces cooking loss and maintains the product's juiciness. Substances such as phosphates and rusk increase the water-holding capacity and are used to produce hams and sausages from PSE pork. However, Fisher et al. (2000c) have shown that the addition of phosphate does not increase pork water absorption, but causes pork products to bind water more completely, thereby minimizing losses during processing.
Genetic factors are among the traits that affect pork quality, and the identification of major genes and molecular markers is a promising approach for improving economic traits, such as pork quality, that are not measurable in breeding animals (Favero, 2002).
Franco et al. (1998) demonstrated that the presence of the n allele led to higher pork drip loss in the semimembranous muscle, thereby reducing its quality. Lundstrom et al. (1995) showed that the PSS gene affected the quality of the longissimus dorsi and resulted in a less tender, less desirable pork. According to Fisher et al. (2000a), a lower initial pH (within 45 min) in Nn pigs, and especially in nn pigs, compromises pork quality because of rapid glycolysis after slaughter.
The use of genomic markers to help in the selection of pork quality is one of the most promising developments in the pig industry. PSE has been known for some time to be associated with variations in the recessive PSS gene (Plastow, 2000), and the availability of a test based on the identification of the causative mutation in the PSS gene described by Fujii et al. (1991) was a key step in marker-assisted screening of pork quality. The test allows breeders to accurately separate all three PSS genotypes, instead of just reactors (nn) from non-reactors (NN and Nn), and has allowed more detailed studies of the effect of this mutation on pork quality.
The aim of this study was to determine the PSS genotypes in F2 pigs derived from divergent crosses and to determine their relationship to pork quality traits.
Materials and Methods
The 596 genotyped F2 pigs were produced by outbreed crossing using 18 commercial females (11 Landrace x Large White and seven Landrace x Large White x Pietrain) with two Brazilian native boars (Piau breed). Both boars and 11 parental females had the NN genotype. The F2 pigs were reared and slaughtered on the Pig Breeding Farm maintained by the Department of Animal Science, Universidade Federal de Viosa, Viosa, Minas Gerais State, Brazil. The pigs were slaughtered at a live weight of 65.0 ± 5.5 kg and were deprived of food for 18 h before slaughter, but had access to fresh water ad libitum. The pigs were electrically stunned (300V/5 s) and bled by cardiac puncture under the left armpit.
For each pig, the pH (pH45) of the longissimus dorsi was measured 45 min postmortem in the left half of the carcass before cooling and in the right half of the carcass after cooling at 4 °C for 24 h in horizontal freezers. Samples of the longissimus dorsi were then obtained to measure other pork quality traits.
Pork quality traits were evaluated in the Meat Laboratory of the Department of Food Technology, Universidade Federal de Viosa, using the procedures described by Benevenuto Junior (2001) for pH 24 h postmortem (pHu), intramuscular fat (IMF), drip, cooking and total loss, shear force, and objective meat color (lightness, redness, yellowness, hue and chroma).
Genotypic analysis was done in the Laboratory of Animal Biotechnology of the Department of Animal Science, Universidade Federal de Viosa. DNA was salt-extracted from white blood cells collected immediately after slaughter using a standard laboratory protocol. The sequence of the ryr-1 gene that contains the C T mutation responsible for triggering PSS (Fujii et al., 1991) was amplified by PCR-RFLP using the primers cited by O'Brien et al. (1993) and generated a 659 bp product.
The amplification mixture contained 1 U of Taq DNA polymerase (Phoneutria), 0.2 M of each primer (forward - 5'-TCCAGTTTGCCACAGGTCCTACCA-3' - and reverse - 5'-TTCACCGGAGTGGAGTCTCTGAG-T-3'), 2 mM MgCl2, 20 mM Tris, pH 8.3, 50 mM KCl, 0.2 mM dNTPs, and 25 ng of genomic DNA in a final volume of 20 mL, according to the standard protocol described by Fujii et al. (1991).
The samples were distributed into previously labeled microtubes containing the reagent mixture described above and were centrifuged at 7,826 g for 10 s to ensure that the samples were at the bottom of each tube. The microtubes were then placed in the 96-sample tray of the thermocycler (MJ-Research PTC-100). The amplification program, modified from Fujii et al. (1991) and Houde et al. (1993), consisted of initial denaturation at 94 °C for 3 min and 35 cycles at 94 °C for 45 s, 68 °C for 1 min and 72 °C for 1 min, with a final polymerization step at 72 °C for 5 min.
Mutation analysis of the samples amplified as described above was done using the restriction enzyme BsiHKA I (New England Biolabs). This enzyme cleaves the 659-bp sequence containing the PSS mutation and generates fragments of 524 and 135 bp in normal homozygotes (NN), fragments of 524, 358, 166 and 135 bp in heterozygotes (Nn), and fragments of 358, 166 and 135 bp in mutant homozygous pigs (nn). After digestion, the reaction products were analyzed on 8% silver nitrate-stained polyacrylamide gels and the pigs were classified as normal homozygotes (NN), heterozygotes (Nn) and recessive homozygotes (nn) according to the size of the DNA fragments.
Statistical analysis of the association of the genotypes with the traits evaluated was done using the SAS General Linear Models (SAS, 1997) program, according to the following model:
Yijkl = m + Gi + Sj + Lk + eijkl
where Yijkl = observed trait in an animal of genotype i, sex j and batch k, m = general mean, Gi = genotype effect (NN or Nn), Sj = sex effect (1 = castrated male and 2 = female), Lk = batch effect (k = 1, 2, 3, 4 and 5) and eijkl = random error.
Results
The RFLP patterns were as expected. Pigs homozygous for the mutation (nn) were characterized by fragments of 358, 166 and 135 bp, normal pigs (NN) showed the 524-bp and the complementary 135-bp fragment, and heterozygous pigs showed fragments of 524, 358, 166 and 135 bp. The NN and Nn genotypes were found in 493 (82.7%) and 103 (17.3%) pigs, respectively. Since only one nn pig was identified, it was not considered in the analysis. These unusual frequencies in F2 crosses were found to be the result of divergent matting patterns in which parental boars were not carriers and the F1 generation was randomly mated regardless of the PSS genotype.
The mean results and number of observations for each pork quality trait in each genotype are shown in Table 1. The traits showing significant differences between the NN and Nn genotypes were pH45 and drip, cooking and total losses. Nn pigs had a higher glycolytic rate postmortem, as indicated by their lower (p < 0.05) pH45 values and higher (p < 0.05) drip, cooking and total losses.
Discussion
The significantly lower pH45 and higher drip, cooking and total losses seen in Nn pigs agreed with other reports in the literature (McPhee and Trout, 1995; Lundstrom et al., 1995; Leach et al., 1996; Monin et al., 1999; Fernandez et al., 2002; Green, 1997; Franco et al., 1998; Miller et al., 1999; Jeremiah et al., 1999; Fisher et al., 2000b) and showed that the presence of the n allele negatively affected pork quality traits by producing greater muscle acidity that led to greater losses during storage and cooking, and produced less juicy pork.
Drip loss, an indicator of the muscle's water holding capacity, is negatively correlated with pH45 (Benevenuto, 2001) and is highly dependent on the initial denaturation of pork myofibrillar proteins.
The lack of a significant difference in objective color indices and tenderness between the Nn and NN genotypes has also been observed by others (Bastos et al., 2001; Fernandez et al., 2002; Miller et al., 1999; Jeremiah et al., 1999). However, discrepancies in pork quality traits have also been reported. Differences in lightness (McPhee and Trout, 1995; Leach et al., 1996; Green, 1997; Bastos et al., 2001), pH24 (Leach et al., 1996; Green, 1997), shear force (Fisher et al., 2000a) and intramuscular fat (Zhang et al., 1992; Leach et al., 1996) between the NN and Nn genotypes have been considered to be indicative of the negative effect of the n allele on protein denaturation and pork quality (Fisher et al., 2000a). According to the latter authors, pork from Nn pigs is less tender and paler than pork from NN pigs because of higher shear force and lightness values (p < 0.05), both of which are associated with a higher incidence of PSE pork as a result of sarcoplasmic protein denaturation and a consequent reduction in pork quality.
In the present study, PSS gene carriers (Nn pigs) had a poorer pork quality, as indicated by their lower pH45 and higher drip, cooking and total losses. The effect of the PSS gene was demonstrable in pigs resulting from divergent crosses, which are generally less susceptible to stress. These results indicate that this gene is one of the main genes to be studied in relation to pork quality.
Acknowledgments
This work was supported by FAPEMIG, CNPq and CAPES.
References
Bastos RG, Federezzi J, Deschamps JC, Cardellino RA and Dellagostin OA (2001) Efeito do gene do estresse suino sobre caracteristicas de quantidade e qualidade de carcaa. Revista Brasileira de Zootecnia 30:37-40.
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