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ANIMAL PRODUCTS |
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* Laboratoire de Zootechnie, Ecole Nationale Vétérinaire de Lyon, 69280 Marcy L’Etoile, France;
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Neuropathology Department, Clinique St Luc, ULB, 1200 Bruxelles, Belgium;
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INSERM U689 Lariboisière, 75475 Paris, France; and
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CEA, 92265 Fontenay-aux-Roses, France
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: halothane • histology • meat quality pig • RN genotype
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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, 2000). Despite the progress to reduce PSE meat in the production lines, numerous studies have been undertaken to find the major risk factors. Many of them concluded that pigs carrying the halothane-sensitivity (n) allele at the HAL/RYR1 locus (Monin et al., 1998; Sellier, 1998; Franck et al., 1999), the RN– allele at the rn locus (Monin et al., 1998; Sellier, 1998; Franck et al., 2000, 2003), or both, present a higher frequency of this defect than normal animals. Nevertheless, the genetic predisposition cannot be considered as the determining factor alone, because this defect is also observed within genetic types free of the HALn and RN– alleles (Franck et al., 1999).
Thus, other risk factors have been evaluated given the likely multiplicity of interwoven mechanisms of PSE meat development. First, it is essential to understand why only some muscles [M. longissimus dorsi (LD) and the M. semimembranosus (SM)] are first involved. According to Lefaucheur et al. (2002), Lefaucheur (2003), and our previous results, it appeared that a high proportion of fibers with a glycolytic metabolism could be partly responsible for the PSE expression within some muscles. Furthermore, a low vascular level could lead to lactic acid trapping into the less vascularized muscles, inducing the acid necrosis. Previously, Sosnicki et al. (1998) and Franck et al. (1999) suspected a probable contribution of a blood flow variation in the development of this defect.
Therefore, the purpose of this study was to compare the fiber typing and capillary network in several affected or nonaffected muscles within different stress-susceptibility genotypes and to provide new insights into the pathophysiology of the PSE meat condition.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Animals
Five groups of pigs were purchased from Porc Hybride Society, kept in a house for experimental animals in the Ecole Nationale Vétérinaire de Lyon, and handled in accordance with national guidelines. Pigs were classified as noncarriers (rn++) or carriers (RN–) at the rn locus mapped on pig chromosome 15 (Milan et al., 1995, 1996; Mariani et al., 1996). Two groups were RN–/rn+, and 3 groups were rn+/rn+; they will be named "+–" and "++," respectively. The different groups were also classified as noncarriers (NN) or carriers of the halothane-sensitivity allele (Nn and nn) at the HAL locus mapped on pig chromosome 6 (Fujii et al., 1991; Otsu et al., 1991). These genetic tests were done according to molecular biology methods (Labogena, Domaine de Vilvert-78352 Jouy en Josas, Cedex, France). These pigs were slaughtered at 100 to 120 kg of BW. They received a general anesthesia (Zoletil 100, tiletamin, 8 mg/kg i.m., and myorelaxation with Nesdonal 1G, thiopental sodium, 20 mg/kg i.v., Alcyon-01706 Miribel Cedex, France) before transport to the autopsy amphitheater and subsequent exsanguination.
Muscle Sampling
Thirty minutes after slaughter, samples were taken from the M. triceps brachii (TB), the LD, the superficial and deep parts of the M. biceps femoris (BF), and the superficial and deep areas of the proximal and median parts of the SM. For each muscle, samples were divided in 2 parts. One part was fixed in formalin, and the other was promptly frozen in isopentane, cooled to its freezing point by liquid N, and stored at –80°C.
Histological Examinations
Histology. Conventional hematoxylin-eosin (HE) (Masson’s method) and a periodic acid-Schiff’s (PAS) staining (Mac Manus’ method) were performed.
Histoenzymology.
Transverse serial sections, 7-mm thick, were cut in a cryostat (2800 Frigocut, Reichert-Jung, Heidelberg, Germany) at –20°C. The conventional myofibrillar ATPase staining after preincubation at pH 4.35 allowed the distinction of 3 fiber types (i.e., black, type I; unstained, type IIa; and gray, type IIb) as defined by Brooke and Kaiser (1970). The cross-sectional area of the fibers differed according to fiber types, and, moreover, the number of the fibers of type I, type IIa, and type IIb varied among the muscles examined. Therefore, because the objective of this study was to correlate the number of each fiber type with the extent of the capillary network within the same defined section, we did not take into account the percentage of fibers of type I, IIa, and IIb. Consequently, for each muscle, 10 cross-sectional areas were photographed at the same magnification (10x) and then examined to count fibers of the different types and to record the average numbers of type I, IIa, and IIb fibers per area and the capillary number per area.
Immunohistochemistry.
Immunohistochemistry was performed on serial, 7-µm thick sections. The sections were briefly incubated for 2 h with the smooth muscle 
-actin antibody (DAKO, Glostrup, Denmark), and the specific binding was revealed by the avidin-biotin-peroxidase complex (Vectastain ABC kit; Vector Laboratories, Bulingame, CA) method. Capillary pericytes and vascular smooth muscle cells are labeled with this antibody. As for the histoenzymology, 10 cross-sectional areas were photographed at the same magnification (10x) for each muscle, then only capillaries, which are the site of nutrient exchange, were counted to determine the average value of the capillary number per area.
Statistical Analysis
For statistical analysis, we pooled the results of values obtained with the samples taken from SM (a proximal superficial sample, a proximal deep sample, a median superficial sample, and a median deep sample) as well as from BF (a superficial sample and a deep sample). Data were used as variables [number of type I, IIa, and IIb fibers; capillaries per area; and the PSE index (PSE-I) value]. The analysis used the GLM procedure (ANOVA, Minitab 13.2, 13 acres in State College, PA). The model included the effects of genotype and of the different muscles studied. The interactions between genotypes (HAL and RN) also were studied. Statistical significance was set at P < 0.05. To simplify the Table 1 data, we noted only the significances due to the PSE-I (see the next paragraph).
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A high number of glycolytic fibers and a low capillary value were mostly observed in the affected muscles. Consequently, to better appreciate the results, a PSE-I was defined. The PSE-I was calculated as follows: the number of type IIb fibers x the total number of fibers per area x the capillary number; this value was then multiplied by 100. The more the PSE-I was increased, the more the muscle was predisposed toward PSE. This synthetic index will be used instead of giving a detailed account of the type IIb fibers and capillary network. However, when PSE-I results had to be explained, details will be presented.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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The muscles from the different genotypes did not show any PSE lesion during the autopsy process.
Histology
Stainings with HE and PAS showed slight well-known lesions (data not shown). First, in the NN+–and Nn+– muscles, a glycogen overloading was observed as well as some lymphocytic infiltrations. Second, nn++ muscles showed minicores or cores. They were seldom noticed in Nn++ muscles. Third, fiber necroses and irregular fibers were frequently observed in nn++ muscles, a little less in Nn++ and NN+–, and rarely seen in Nn+–.
Immunohistochemistry
The smooth muscle -actin showed variations in staining intensity. The Nn++, Nn+–, and nn muscle pericytes and vascular smooth muscle cells often presented a heterogeneous staining even in the arterioles compared with the homogeneous staining of NN++ and NN+– vascular smooth muscle cells (Figure 1, panel B). The capillary network in TB was dramatically increased (P < 0.001) compared with the other muscles (Figure 1, panel A). Intriguingly, in the BF of the nn++ group, the capillary network varied up to 2-fold depending on the superficial region compared with the deep part of this muscle. It was not noticed in the other groups and in the SM among the 4 different sampled areas.
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Differences in fiber types were observed in histoenzymology (Figure 1
, panel C) and recorded, as well as the capillary number obtained with the smooth muscle -actin labeling. A histogram emphasized the results (Figure 2). There was no obvious difference in fiber size within the 4 studied muscles, because there was a similar total fiber number per area. As TB corresponds to the most often spared muscle during and after a stress, it was used as a muscle of reference. In TB, mean values of fiber typing were 32 ± 17 for type I, 18.5 ± 11 for type IIa, and 73 ± 34 for type Iib, respectively. The capillary number per area was 40 ± 15. The other muscles (LD, BF, and SM) harbored a significant increase in the number of type IIb fibers (P < 0.001; Figure 2). The capillary network value per area of LD, BF, and SM significantly decreased compared with TB (P < 0.001), especially in LD and SM showing 50% less capillary network than in TB. Finally, the PSE-I followed the increase in fiber type IIb number in these muscles (LD, BF, and SM), but this increase was more pronounced, because capillary number was also taken into account. So the PSE-I showed an increase (P < 0.001) from 1.9 ± 1.3 in TB to 5 ± 3.7 in LM, 5.8 ± 6.8 in BF, and 11.6 ± 11.6 in SM, respectively.
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We did not find any interaction between genetic types (HAL and RN) with the studied phenotypes. Conversely, studies of the different genotypes brought some interesting points.
HAL Effect on rn++.
First, the HAL effect on rn++ was checked. This part concerns the 3 groups NN++, Nn++, and nn++, in which 4 muscles were studied (Table 1). Apart from a slight decrease in the SM, the total number of fibers was not significantly different among the muscles independently from the genotypes. There was no obvious difference between the NN++ and Nn++ groups in all studied muscles. Type IIb fiber number did not exhibit a significant difference between the different genotypes in the muscles studied compared with the muscle of reference (TB) apart from the LD, which showed a increase (P < 0.001) in the nn++ genotype (Table 1). A significant reduced capillary network (P < 0.001) was noticed in LD, BF, and SM of NN++ and Nn++ genotypes compared with their TB and in LD and SM of the nn genotype compared with its TB and BF. The biggest decrease (around 50%) in capillary network was found in the LD of the nn++ group. The PSE-I value of TB was low in all genetic types but significantly increased in the nn++ group compared with the NN++ group (P < 0.001) and the Nn++ group (P < 0.01). In LD, BF, and SM, the PSE-I values doubled or more in all different genotypes, apart from the PSE-I value of BF in the nn++ group. Nevertheless, in nn++ muscles, all PSE-I values were clearly over the double of the PSE-I values observed in the NN++ and Nn++ TB muscles.
RN-rn+ Effect on NN and Nn. Second, the RN-rn+ effect on NN and Nn was checked. In this part, only TB and LD were studied, because the posterior part of these animals was used in other experimentation concerning the repartition of large vessels.
). These results bear out our first histological examination of muscles of the Nn+– genotype, in which we already noticed fewer necrosis features and irregular fibers. ). ). |
DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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, 2000). On the other hand, the measurements of the color with a Minolta Chromameter CR-300 (Minolta, Osaka, Japan) give excellent results concerning the prediction of the slicing productivity (P. Figwer, personal communication). Two muscles are first and foremost involved: the LD in the loin and the SM in the ham. These 2 carcass parts correspond to the meat pieces that add the most value to the slicing industry. Therefore, the PSE meat has a huge negative effect on the manufacturers’ productivity. This emphasizes the absolute necessity to do new research, and this study may be one of the first demonstrations of muscle vulnerability due to a structural predisposition in some of them.
The first point emphasized in this study was the obvious decrease in capillary value between the TB, which is mostly spared, compared with the other 3 muscles usually involved during stress. As we know, the capillary network is directly proportional to the number of type I fibers, which are oxidative fibers (Ruusunen and Puolanne, 2004), and we showed that the TB contained the higher number of type I fibers compared with the 3 other muscles. This finding justified the choice of the TB as the control muscle, because it is seldom involved. The second point was the obvious increase in glycolytic type IIb fibers in the 3 other studied muscles. The more the muscle contains glycolytic fibers, the more lactic acid could be increased in the event of stress. Moreover, according to our results, the more the muscle contained glycolytic fibers, the less the capillary network was developed, resulting in a very low level in capillary network exchanges. Consequently, lactic acid might be trapped in these muscles with a reduced capillary network. Capillaries are the only sites of exchanges, and for this 1 reason, capillaries were counted in this study and not the arterioles. The PSE-I value was likely to bring new information, because the numbers of capillary networks and glycolytic fibers were taken into account. Indeed, in the part taking all genotypes together, the PSE-I of each muscle seemed worth considering, because it gave an accurate representation of the ground observations. The SM harbored the greatest PSE-I (11.6), and the LD and BF also harbored a great PSE-I (5 to 5.8, respectively) when the TB harbored the lowest (1.9). This index put the TB in the shelter from easy PSE meat condition. One could be tempted to follow the same explanation concerning the meat vulnerability in the group carrying the halothane-sensitivity allele (n). But the second part of our study showed that if the PSE-I followed a similar curve in all studied genotypes, they were not systematically lower in all muscles of the NN++ genotype compared with the Nn++ and nn++ genotypes. The NN genotype exhibited lower PSE-I (P < 0.001) in TB and LD compared with nn++. Conversely, it was of interest to note that in the nn++ genotype, BF and SM muscles contained a slightly more developed capillary network than in the NN++ and Nn++ genotypes. As a consequence, BF and SM muscles in the nn++ genotype presented greater PSE-I (3.2 to 5.4, respectively), nevertheless lower (P < 0.001) than BF and SM muscle PSE-I in NN++ and Nn++ genotypes (9.1 to 16.1 and 4.9 to 12.4, respectively). Despite this fact, the NN++ genotype is seldom affected. One has to keep in mind that a well-known risk factor is added in the other genotypes with the presence of a mutation in the HAL/RYR1 gene. The first RYR1 mutation, an Arg615Cys detected by Fujii et al. (1991) and Otsu et al. (1991), corresponds to the most frequent human mutation, an Arg614Cys substitution (Gillard et al., 1991). This mutation alters the RYR1 function, which is a Ca release channel. The point mutation Arg615Cys in the Ca2+ release channel of skeletal sarcoplasmic reticulum is responsible for hypersensitivity to caffeine and halothane in malignant hyperthermia, leading to hypercontraction and muscle necrosis (Otsu et al., 1994). Functional tests, so far only performed with porcine muscle in sarcoplasmic reticulum vesicles, have shown that Ca regulation is disturbed. Lower Ca concentrations activate the channel to a higher than normal level, and higher than normal Ca concentrations are required to inhibit the channel (Mickelson and Louis, 1996). This factor is likely to be determined on predisposed muscles such as LD, BF, and MS, which contained a high type IIb fiber proportion and a reduced capillary network compared with the less-affected TB. Our hypothesis concerning the meat vulnerability also fits exactly over the NN+– group; nevertheless, this work added a new argument to previous studies. In agreement with previous authors (Lebret et al., 1999; Dépreux et al., 2000), the NN+– group showed a decrease in type IIb fiber size in LD compared with TB. Because the mean value of all fibers per area was increased (72 in TB to 112 in LD), this resulted in a decrease in fiber area for most of the fibers. Previously, Lebret et al. (1999) had expected that LD contained less glycogen because of the decreased relative area of glycolytic fibers in LD. Therefore, they incriminated a default in glycogen metabolism in white muscles of RN– carriers. Our study showed that RN carriers exhibited both a decrease in type IIb fiber size and an increase in the number of type IIb fibers per area in LD compared with TB. So the increased number of type IIb fibers per area in LD, added to the glycogen overloading observed with the PAS staining (obviously due to a glycogen metabolism defect), elicited the dramatic increase in glycogen content as assessed by glycolytic potential and a decrease in ultimate pH, DM, and protein content previously observed by Lebret et al. (1999). The single presence of a glycogen overloading in muscle predisposed this muscle to produce more lactic acid. In Rn+–, there was more glycogen in all muscles, and some muscles presented more type IIb fibers and a reduced capillary network. Consequently, more lactic acid was likely to be trapped in these muscles. At least we noticed that the Nn+– group harbored a slightly lower PSE-I compared with the NN+– group in the LD, confirming our previous standard histological observations that showed less fiber lesions in the Nn+–group. As a matter of fact, everything is based on the presence of more type IIb fibers and less capillaries in LD of NN+– compared with LM of Nn+–. Therefore, it resulted in a higher PSE-I in the NN+– group compared with the Nn+– group. The PSE-I value seemed to correlate perfectly with the histological observation of less fiber lesions in Nn+– muscle compared with NN+– muscle. Intriguingly, the PSE-I emphasizes the fact that the presence of an allele n at the HAL locus added to the RN– allele at the rn locus was likely to be a positive compensatory factor and not a cumulative negative factor, as it was previously thought (Le Roy et al., 2000). In the future, interactions between genetic types could be particularly interesting to observe even if we do not find significant interaction with the currently studied phenotypes. These interactions would force us not to independently consider the loci (HAL and RN), and they could also open new ways for research of unknown physiopathologic mechanisms.
Finally, the results summarized in the first histogram taking all genotypes together allowed a better understanding about the vulnerability of some muscles, which usually show a PSE aspect. The PSE-I highlighted these findings and inversely correlated perfectly with the pork meat vulnerability. Indeed, SM and BF are the most affected muscles in the ham as well as the LD in the loin, even in nonsusceptible pigs (NN++). All these muscles harbored a dramatic high PSE-I value clearly over the double of the PSE-I value in the TB of NN++. Conversely, TB harbored low PSE-I apart from the nn++ group, and actually it is in the nn++ group that one can observe a PSE-type destruction of all the muscles, even in the TB. These new data suggest that PSE-I could be a good index to value the vulnerability of some muscles with a probable cutoff around 2. This work also gives new arguments to previous studies (Sellier and Monin, 1994; Larzul et al., 1997; Sellier, 1998; Lebret et al., 1999; Franck et al., 1999, 2000, 2002; Le Roy et al., 2000; Laville et al., 2005) to elucidate the vulnerability of some muscles with a close correlation to a high level of type IIb fibers and a low level in capillary networks for all the currently studied genotypes. However, added to the marked vulnerability of muscles harboring a PSE-I greater than 2, there are other dramatic risk factors in the presently studied genotypes. These are an increase in glycogen content in the RN– carriers affecting all muscles and a hypersensitivity to the stress in Nn and nn genotypes.
These new data highlighted the PSE meat pathophysiology and explained, in part, the fact that in all studied genotypes, the first muscles to be involved are the LD and SM and the BF, even though all the muscles could be involved. We must mention that at the end of this study, we detected modifications within vessels in the Nn++ and nn++ groups. Capillaries and arterioles showed heterogeneous HE staining with the presence of cytoplasmic microvacuoles, as well as a irregular labeling with the smooth muscle -actin antibody (Figure 1, panel B), likely because of an edematous aspect of pericytes and vascular smooth muscle cells. Future structural studies at higher resolutions are needed to refine these changes and to substantiate a proposed hypothesis concerning modifications of vessel wall function, likely the permeability, vasomotricity, or both in the n carriers. Some authors (Sosnicki et al., 1998; Franck et al., 1999) have already suspected a vascular factor, because pigs that carry 1 or 2 copies of the n allele are leaner and generally more heavily muscled (Christian and Rothschild, 1981). So a modification of the permeability is likely to be one of the factors playing a part in the much sought-after meat quality found in pigs carrying 1 or 2 copies of the n allele at the HAL locus.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: m.franck{at}vet-lyon.fr
Received for publication March 28, 2006. Accepted for publication September 25, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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